High-risk human papillomavirus detection

ABSTRACT

This invention provides compositions and methods for detecting HPV in a sample. This invention also provides related kits, systems, and computers.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 12/332,164,filed on Dec. 10, 2008, which is a divisional of application Ser. No.11/119,343, filed on, Apr. 29, 2005, now U.S. Pat. No. 7,482,142, whichclaims the benefit of the U.S. Provisional Application No. 60/568,934,filed on May 7, 2004, the disclosures of which are incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology andnucleic acid chemistry. The invention provides methods and reagents fordetecting human papillomavirus and accordingly, also relates to thefields of medical diagnostics and prognostics.

BACKGROUND OF THE INVENTION

Human papillomaviruses (HPVs) are a group of epitheliotropic virusescharacterized by a circular, double-stranded DNA genome. Different HPVtypes infect the skin or the mucosa of the respiratory and anogenitaltract, and correlate with benign and malignant neoplasia of cutaneousand mucosal epithelia. To illustrate, HPV DNA is found in essentiallyall cases of cervical carcinoma and in many precursor lesions. Cervicalcarcinoma is currently second only to breast cancer as the mostprevalent malignancy in women worldwide.

In excess of 100 different HPV types, which are numbered inchronological order of isolation, have been characterized to date. AnHPV genome is currently classified as a new type if it has less than 90%DNA sequence homology to other HPVs in the L1 open reading frame.Further, based on the induced benign, premalignant or malignant lesionsof the cervix, anogenital HPV is divided into low-risk (including, e.g.,types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) and high-risk(including, e.g., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,68, 73, and 82) types, respectively. See, e.g., Munoz et al. (2003)“Epidemiologic classification of human papillomavirus types associatedwith cervical cancer,” N. Engl. J. Med. 348(6):518-527, which isincorporated by reference. The genome of low-risk HPV types typicallyremains episomal, while the circular ds-DNA genome of high-risk HPVtypes may integrate into the human genome as part of carcinogenesis.Moreover, high-risk HPV types account for more than 80% of all invasivecervical cancers. As a consequence, the detection and identification ofparticular HPV types present in patient samples provides significantdiagnostic and prognostic information.

Various approaches to the diagnosis of HPV infections have beenattempted. Culture-based techniques have generally proven to beunfeasible. Immunoassays directed at HPV detection are typically limitedby insufficient sensitivity and specificity. The most successful methodsof diagnosing HPV infections typically involve nucleic acidhybridization-based assays with or without the amplification of targetHPV DNA sequences.

Many hybridization-based diagnostic tests lack sufficient specificity todifferentiate between high- and low-risk HPV types or to distinguishbetween various high-risk HPV types. This can lead to biased assayresults, including false positives. One consequence of such misdiagnosismay be the administration of an inappropriate course of treatment to apatient.

SUMMARY OF THE INVENTION

The present invention provides methods and reagents for the rapiddetection of HPV. For example, the invention provides cross-reactiveoligonucleotides for the detection of various high-risk HPVs, includingHPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58. In addition tocompositions and reaction mixtures, the invention also relates to kitsand systems for detecting these pathogenic agents, and to relatedcomputer and computer readable media.

In one aspect, the invention provides an oligonucleotide consisting of anucleic acid with a sequence selected from the group consisting of: SEQID NOS: 1-31 and complements thereof. In another aspect, the inventionprovides an oligonucleotide comprising a nucleic acid with a sequenceselected from the group consisting of: SEQ ID NOS: 1-31 and complementsthereof, which oligonucleotide consists of 100 or fewer nucleotides. Instill another aspect, the invention relates to an oligonucleotidecomprising a nucleic acid having at least 90% sequence identity to oneof SEQ ID NOS: 1-31 or a complement thereof, which oligonucleotideconsists of 100 or fewer nucleotides. In some embodiments, the nucleicacid has at least 95% sequence identity to one of SEQ ID NOS: 1-31 orthe complement thereof. In certain embodiments, the nucleic acidcomprises at least one modified nucleotide substitution (e.g., betweenabout two and about 20 modified nucleotide substitutions).

In another aspect, the invention relates to a composition comprising asample (e.g., derived from a subject) and at least one oligonucleotidethat comprises a nucleic acid with a sequence selected from the groupconsisting of: SEQ ID NOS: 1-31, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1-31, and complements of SEQ ID NOS: 1-31 and thevariant, which oligonucleotide consists of 100 or fewer nucleotides.

The oligonucleotides of the compositions described herein are providedin various formats. In certain embodiments, for example, at least one ofthe oligonucleotides is in solution. In some embodiments, a solidsupport comprises the oligonucleotide. Optionally, the oligonucleotideis non-covalently or covalently attached to the solid support. Exemplarysolid supports utilized in these embodiments are optionally selectedfrom, e.g., a plate, a microwell plate, a bead, a microbead, a fiber, awhisker, a comb, a hybridization chip, a membrane, a single crystal, aceramic layer, a self-assembling monolayer, and the like. In certainembodiments, a linker attaches the oligonucleotide to the solid support.The linker is optionally selected from, e.g. an oligopeptide, anoligonucleotide, an oligopolyamide, an oligoethyleneglycerol, anoligoacrylamide, an alkyl chain, and/or the like. To further illustrate,the oligonucleotide is optionally bovine serum albumin-conjugated. Insome embodiments, a cleavable attachment attaches the oligonucleotide tothe solid support. For example, the cleavable attachment is typicallycleavable by heat, an enzyme, a chemical agent, electromagneticradiation, or the like.

In another aspect, the invention provides a method of determining apresence of at least one high-risk human papillomavirus (HPV) type in asample. The method includes (a) contacting nucleic acids and/oramplicons thereof from the sample with at least one oligonucleotide thatcomprises a nucleic acid with a sequence selected from the groupconsisting of: SEQ ID NOS: 1-31 and complements of SEQ ID NOS: 1-31,which oligonucleotide consists of 100 or fewer nucleotides. The methodalso includes (b) monitoring binding between the nucleic acids and/oramplicons thereof, and the oligonucleotide, in which detectable bindingbetween the nucleic acids and/or amplicons thereof, and theoligonucleotide, determines the presence of the high-risk HPV type inthe sample.

In still another aspect, the invention relates to a method ofdetermining a presence of at least one high-risk human papillomavirus(HPV) type in a sample. The method includes (a) contacting nucleic acidsand/or amplicons thereof from the sample with at least oneoligonucleotide that comprises a nucleic acid having at least 90%sequence identity to one of SEQ ID NOS: 1-31 and complements of SEQ IDNOS: 1-31, which oligonucleotide consists of 100 or fewer nucleotides.Typically, the sample is derived from a human subject. The method alsoincludes (b) monitoring binding between the nucleic acids and/oramplicons thereof, and the oligonucleotide, in which detectable bindingbetween the nucleic acids and/or amplicons thereof, and theoligonucleotide, determines the presence of the high-risk HPV type(e.g., HPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58) in the sample.In certain embodiments, for example, (b) comprises monitoring bindingbetween the oligonucleotide and the nucleic acid and/or ampliconsthereof, if any, under stringent hybridization conditions. Optionally,the method comprises repeating (a) and (b) at least once using at leastone additional sample and comparing the binding between the nucleicacids and/or amplicons thereof, and the oligonucleotide, of (b) with atleast one repeated (b). In some embodiments, the nucleic acids and/oramplicons thereof and the oligonucleotide are contacted in solution. Incertain embodiments, a solid support comprises the nucleic acids and/oramplicons thereof, whereas in others, a solid support comprises theoligonucleotide.

In certain embodiments, at least one segment of the nucleic acids isamplified prior to or during (a) using at least one nucleic acidamplification technique to produce the amplicons and (b) comprisesmonitoring the binding between the nucleic acids and/or ampliconsthereof, and the oligonucleotide, during or after amplification. Inthese embodiments, the nucleic acid amplification technique utilizes atleast one primer nucleic acid that comprises at least one labelingmoiety.

In some embodiments, the nucleic acids and/or amplicons thereof, and/orthe oligonucleotide comprises at least one labeling moiety (e.g., afluorescent labeling moiety, etc.) and/or at least one quencher moiety.In these embodiments, (b) comprises detecting a detectable signalproduced by the labeling moiety. Optionally, (b) comprises: (i)amplifying a detectable signal produced by the labeling moiety toproduce an amplified signal, and (ii) detecting the amplified signal.

In another aspect, the invention provides a kit that includes (a) atleast one oligonucleotide that comprises a nucleic acid with a sequenceselected from the group consisting of: SEQ ID NOS: 1-31, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 1-31, and complements of SEQ ID NOS: 1-31and the variant, which oligonucleotide consists of 100 or fewernucleotides. The kit also includes (b) instructions for determining apresence of at least one high-risk human papillomavirus (HPV) type(e.g., HPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58) in a sample bymonitoring binding between nucleic acids and/or amplicons thereof fromthe sample and the oligonucleotide. In some embodiments, theoligonucleotide is in solution. In other embodiments, a solid support(e.g., a plate, a microwell plate, a bead, a microbead, a fiber, awhisker, a comb, a hybridization chip, a membrane, a single crystal, aceramic layer, a self-assembling monolayer, etc.) comprises theoligonucleotide. Optionally, the kit further includes (c) at least onecontainer for packaging the oligonucleotide and/or the instructions. Insome embodiments, the kit further comprises instructions for obtainingsamples.

In certain embodiments, the kit further includes at least one primernucleic acid that is at least partially complementary to at least onesegment of an L1 region of the high-risk HPV type. In these embodiments,the kit optionally further includes instructions for amplifying one ormore segments of the L1 region with the primer nucleic acid, at leastone nucleotide incorporating biocatalyst, and one or more nucleotides.The primer nucleic acid optionally comprises at least one labelingmoiety. In some of these embodiments, the kit further includes at leastone nucleotide incorporating biocatalyst (e.g., a polymerase, a ligase,etc.) and/or one or more nucleotides.

In one aspect, the invention provides a system for detecting at leastone high-risk human papillomavirus (HPV) type (e.g., HPV31, HPV33,HPV35, HPV52, HPV56, and/or HPV58) in a sample. The system includes (a)at least one oligonucleotide that comprises a nucleic acid with asequence selected from the group consisting of: SEQ ID NOS: 1-31, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 1-31, and complementsof SEQ ID NOS: 1-31 and the variant, which oligonucleotide consists of100 or fewer nucleotides. The system also includes (b) at least onedetector that detects binding between nucleic acids and/or ampliconsthereof from the sample and the oligonucleotide. In addition, the systemalso includes (c) at least one controller operably connected to thedetector, which controller comprises one or more instructions sets thatcorrelate the binding detected by the detector with a presence of thehigh-risk HPV type in the sample. In certain embodiments, at least onecontainer or solid support comprises the oligonucleotide. In some ofthese embodiments, the system further includes (d) at least one thermalmodulator operably connected to the container or solid support tomodulate temperature in the container or on the solid support, and/or(e) at least one fluid transfer component that transfers fluid to and/orfrom the container or solid support.

In another aspect, the invention provides a system that includes (a)computer or computer readable medium comprising a data set thatcomprises at least one character string that corresponds to at least onesequence selected from the group consisting of: SEQ. ID NOS. 1-31 andcomplements thereof. The system also includes (b) an automaticsynthesizer coupled to an output of the computer or computer readablemedium, which automatic synthesizer accepts instructions from thecomputer or computer readable medium, which instructions directsynthesis of one or more nucleic acids that correspond to one or morecharacter strings in the data set.

The oligonucleotides, including those provided for in the compositions,methods, kits, and systems described herein, include variousembodiments. In some embodiments, for example, variants have at least90% sequence identity to one of SEQ ID NOS: 1-31 and complementsthereof, whereas in others, the variants have at least 95% sequenceidentity to one of SEQ ID NOS: 1-31 and complements thereof. In certainembodiments, the sequence of the nucleic acid comprises at least onemodified nucleotide substitution, e.g., such that the oligonucleotidecomprises a substantially identical variant having at least 90% sequenceidentity to one of SEQ ID NOS: 1-31. For example, the sequence of thenucleic acid optionally comprises at least one nucleotide mismatch witha substantially complementary sequence in an L1 region of at least onehigh-risk human papillomavirus (HPV) type. In these embodiments, themodified nucleotide substitution is typically positioned proximal to thenucleotide mismatch. To illustrate, the sequence of the nucleic acidoptionally comprises between about two and about 20 modified nucleotidesubstitutions. The modified nucleotide substitution generally modifies amelting temperature (T_(m)) of the oligonucleotide by at least about0.2° C. relative to a T_(m) of a corresponding unmodifiedoligonucleotide. Optionally, the modified nucleotide is selected from,e.g., a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a 7-deaza-dG, an N4-ethyl-dC, anN6-methyl-dA, or the like. In some embodiments, the oligonucleotideincludes at least one labeling moiety and/or at least one quenchermoiety. For example, the labeling moiety optionally comprises bovineserum albumin, a fluorescent dye, a weakly fluorescent label, anon-fluorescent label, a colorimetric label, a chemiluminescent label, abioluminescent label, an antibody, an antigen, biotin, a hapten, amass-modifying group, a radioisotope, an enzyme, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing a representative example system fordetecting HPV in a sample.

FIG. 2 is a block diagram showing a representative example systemincluding a computer and a computer readable medium in which variousaspects of the present invention may be embodied.

FIG. 3 is a graph showing the specificity of the A3X5X oligonucleotideseries for various HPV targets.

FIG. 4 is a melting curve for perfect match sequences.

FIG. 5 is a melting curve for single mismatch sequences.

DETAILED DESCRIPTION I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularoligonucleotides, methods, compositions, kits, systems, computers, orcomputer readable media, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Further, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. In describing and claiming the present invention, thefollowing terminology and grammatical variants will be used inaccordance with the definitions set forth below.

A “5′-nuclease probe” refers to an oligonucleotide that comprises atleast two labels and emits radiation of increased intensity after one ofthe two labels is cleaved or otherwise separated from theoligonucleotide. In certain embodiments, for example, a 5′-nucleaseprobe is labeled with two different fluorescent dyes, e.g., a 5′terminus reporter dye and the 3′ terminus quencher dye or moiety. Insome embodiments, 5′-nuclease probes are labeled at one or morepositions other than, or in addition to, terminal positions. When theprobe is intact, energy transfer typically occurs between the twofluorophores such that fluorescent emission from the reporter dye isquenched. During an extension step of a polymerase chain reaction, forexample, a 5′-nuclease probe bound to a template nucleic acid is cleavedby the 5′ nuclease activity of, e.g., a Taq polymerase such that thefluorescent emission of the reporter dye is no longer quenched.Exemplary 5′-nuclease probes are described in, e.g., U.S. Pat. No.5,210,015, entitled “HOMOGENEOUS ASSAY SYSTEM USING THE NUCLEASEACTIVITY OF A NUCLEIC ACID POLYMERASE,” issued May 11, 1993 to Gelfandet al., U.S. Pat. No. 5,994,056, entitled “HOMOGENEOUS METHODS FORNUCLEIC ACID AMPLIFICATION AND DETECTION,” issued Nov. 30, 1999 toHiguchi, and U.S. Pat. No. 6,171,785, entitled “METHODS AND DEVICES FORHEMOGENEOUS NUCLEIC ACID AMPLIFICATION AND DETECTOR,” issued Jan. 9,2001 to Higuchi, which are each incorporated by reference.

The term “alteration” refers to a change in a nucleic acid sequence,including, but not limited to, a substitution, an insertion, and/or adeletion. For example, a variant nucleic acid typically comprises one ormore alterations relative to a corresponding non-variant nucleic acid.

An “amplicon” refers to a molecule made by copying or transcribinganother molecule, e.g., as occurs in transcription, cloning, and/or in apolymerase chain reaction (“PCR”) (e.g., strand displacement PCRamplification (SDA), duplex PCR amplification, etc.) or other nucleicacid amplification technique. Typically, an amplicon is a copy of aselected nucleic acid (e.g., a template or target nucleic acid) or iscomplementary thereto.

An “amplification reaction” refers to a primer initiated replication ofone or more target nucleic acid sequences or complements thereto.

An “amplified signal” refers to an increased detectable signal that canbe produced in the absence of, or in conjunction with, a nucleic acidamplification reaction. Exemplary signal amplification techniques aredescribed in, e.g., Cao et al. (1995) “Clinical evaluation of branchedDNA signal amplification for quantifying HIV type 1 in human plasma,”AIDS Res Hum Retroviruses 11(3):353-361, and in U.S. Pat. No. 5,437,977to Segev, U.S. Pat. No. 6,033,853 to Delair et al., and U.S. Pat. No.6,180,777 to Horn, which are each incorporated by reference.

An “array” refers to an assemblage of elements. The assemblage can bespatially ordered (a “patterned array”) or disordered (a “randomlypatterned” array). The array can form or comprise one or more functionalelements (e.g., a probe region on a microarray) or it can benon-functional.

The term “attached” or “conjugated” refers to interactions and/or statesincluding, but not limited to, covalent bonding, ionic bonding,chemisorption, physisorption, and combinations thereof. In certainembodiments, for example, oligonucleotides are attached to solidsupports. In some of these embodiments, an oligonucleotide is conjugatedwith biotin (i.e., is biotinylated) and a solid support is conjugatedwith avidin such that the oligonucleotide attaches to the solid supportvia the binding interaction of, e.g., biotin and avidin.

A “character” when used in reference to a character of a characterstring refers to a subunit of the string. In one embodiment, thecharacter of a character string encodes one subunit of an encodedbiological molecule. Thus, for example, where the encoded biologicalmolecule is a polynucleotide or oligonucleotide, a character of thestring encodes a single nucleotide.

A “character string” is any entity capable of storing sequenceinformation (e.g., the subunit structure of a biological molecule suchas the nucleotide sequence of a nucleic acid, etc.). In one embodiment,the character string can be a simple sequence of characters (letters,numbers, or other symbols) or it can be a numeric or codedrepresentation of such information in tangible or intangible (e.g.,electronic, magnetic, etc.) form. The character string need not be“linear,” but can also exist in a number of other forms, e.g., a linkedlist or other non-linear array (e.g., used as a code to generate alinear array of characters), or the like. Character strings aretypically those which encode oligonucleotide or polynucleotide strings,directly or indirectly, including any encrypted strings, or images, orarrangements of objects which can be transformed unambiguously tocharacter strings representing sequences of monomers or multimers inpolynucleotides, or the like (whether made of natural or artificialmonomers).

The term “cleavage” in the context of solid supports refers to a processof releasing a material or compound from a solid support to permitanalysis of the compound by solution-phase methods. See, e.g., Wells etal. (1998) “Cleavage and Analysis of Material from Single Resin Beads,”J. Org. Chem. 63:6430, which is incorporated by reference. In certainother contexts, such as 5′-nuclease assays or reactions, 5′-nucleaseprobes can be cleaved by the nuclease activity associated with variousnucleic acid polymerases typically utilized in those reactions.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

A “composition” refers to a combination of two or more differentcomponents. In certain embodiments, for example, a composition includesa solid support that comprises one or more oligonucleotides, e.g.,covalently or non-covalently attached to a surface of the support. Inother embodiments, a composition includes one or more oligonucleotidesin solution.

Two nucleic acids “correspond” when they have substantially identical orcomplementary sequences. In certain embodiments, for example, twonucleic acids correspond to one another when the sequence of one nucleicacid is derived naturally or artificially from the other. To furtherillustrate, a substantially identical variant of a nucleic acid includesone or more modified nucleotide substitutions relative to acorresponding unmodified nucleic acid in some embodiments of theinvention.

An oligonucleotide is “cross-reactive” when it is capable of selectivelyhybridizing to two or more target nucleic acids under suitableconditions to permit detection of those target nucleic acids.

The term “deletion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is removed from thenucleic acid sequence, e.g., from a 5′-terminus, from a 3′-terminus,and/or from an internal position of the nucleic acid sequence.

The term “derivative” refers to a chemical substance relatedstructurally to another substance, or a chemical substance that can bemade from another substance (i.e., the substance it is derived from),e.g., through chemical or enzymatic modification. To illustrate,oligonucleotides are optionally conjugated with biotin or a biotinderivative. To further illustrate, one nucleic acid can be “derived”from another through processes, such as chemical synthesis based onknowledge of the sequence of the other nucleic acid, amplification ofthe other nucleic acid, or the like.

The term “detectably bind” and “detectable binding” refers to bindingbetween at least two molecular species (e.g., a probe nucleic acid and atarget nucleic acid) that is detectable above a background signal (e.g.,noise) using one or more methods of detection.

Nucleic acids are “extended” or “elongated” when additional nucleotides(or other analogous molecules) are incorporated into the nucleic acids.For example, a nucleic acid is optionally extended by a nucleotideincorporating biocatalyst, such as a polymerase that typically addsnucleotides at the 3′ terminal end of a nucleic acid.

An “extended primer nucleic acid” refers to a primer nucleic acid towhich one or more additional nucleotides have been added or otherwiseincorporated (e.g.; covalently bonded thereto).

A “genotype” or “type” refers to all or part of the genetic constitutionof a virus, a cell, or subject. For example, the cross-reactiveoligonucleotides of the invention bind to multiple high-risk HPV types(selected from, e.g., HPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58)under selected conditions.

The term “high risk human papillomavirus type” or “high risk HPV type”refers types of anogenital HPV that are considered to be carcinogenic.Examples of high risk HPV types include 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 68, 73, and 82. HPV classification is also described in,e.g., Munoz et al. (2003) “Epidemiologic classification of humanpapillomavirus types associated with cervical cancer,” N. Engl. J. Med.348(6):518-527, which is incorporated by reference.

The term “human papillomavirus” or “HPV” refers to a double-stranded DNAvirus of the family Papovaviridae, genus Papillomavirus that infectshuman hosts. To date, over 100 types of HPV have been identified.Additional general description of HPV is provided in, e.g., zur Hausen(2002) “Papillomaviruses and cancer: from basic studies to clinicalapplication,” Nat Rev Cancer. 2(5):342-350, Stern et al. (Eds.), HumanPapillomaviruses and Cervical Cancer: Biology and Immunology, OxfordUniversity Press (1994), Sterling et al. (Eds.), Human Papillomaviruses.Clinical and Scientific Advances, Arnold Publications Series, OxfordUniversity Press (2002), Guo, Molecular Mechanisms in CervicalCarcinogenesis Studies of Clonality, Somatic Genetic Alterations andHuman Papillomavirus Variants in Cervical Pre-Invasive and InvasiveCancer, Uppsala University Press (2000), and Syrjanen et al.,Papillomavirus Infections in Human Pathology, John Wiley & Sons, Inc.(1999), which are each incorporated by reference. See also, the HPVSequence Database provided on the world wide web at hpv-web.lanl.gov asof May 7, 2004.

The term “human papillomavirus nucleic acid” or “HPV nucleic acid”refers to a nucleic acid that is derived or isolated from a humanpapillomavirus and/or an amplicon of such a nucleic acid.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization assays or experiments, such as nucleic acid amplificationreactions, Southern and northern hybridizations, or the like, aresequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.

For purposes of the present invention, “highly stringent” hybridizationand wash conditions are selected to be at least about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the test sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids on a filter in a Southern or northern blotis 50% formalin with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of stringent wash conditions isa 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001), which is incorporated byreference, for a description of SSC buffer). Often the high stringencywash is preceded by a low stringency wash to remove background probesignal. An example low stringency wash is 2×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 5× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

Comparative hybridization can be used to identify nucleic acids of theinvention.

In particular, detection of stringent hybridization in the context ofthe present invention indicates strong structural similarity to, e.g.,the nucleic acids provided in the sequence listing herein. For example,it is desirable to identify test nucleic acids that hybridize to theexemplar nucleic acids herein under stringent conditions. One measure ofstringent hybridization is the ability to detectably hybridize to one ofthe listed nucleic acids (e.g., nucleic acids with sequences selectedfrom SEQ ID NOS: 1-31 and complements thereof) under stringentconditions. Stringent hybridization and wash conditions can easily bedetermined empirically for any test nucleic acid.

For example, in determining highly stringent hybridization and washconditions, the stringency of the hybridization and wash conditions aregradually increased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents such as formalin in the hybridizationor wash), until a selected set of criteria is met. For example, thestringency of the hybridization and wash conditions are graduallyincreased until an oligonucleotide consisting of or comprising one ormore nucleic acid sequences selected from SEQ ID NOS: 1-31 andcomplementary oligonucleotide sequences thereof, binds to a perfectlymatched complementary target (again, a nucleic acid comprising one ormore nucleic acid sequences selected from SEQ ID NOS: 1-31 andcomplementary oligonucleotide sequences thereof), with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe oligonucleotide to a non-target nucleic acid. In this case,non-target nucleic acids are those from low-risk HPV types or organismsother than HPV. Examples of such non-target nucleic acids include, e.g.,the L1 region of HPV types 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 57,64, 66, 67, and 70. Additional such sequences can be identified in,e.g., GenBank® by one of skill in the art.

The detection of target nucleic acids which hybridize to the nucleicacids represented by SEQ ID NOS: 1-31 and complements thereof under highstringency conditions are a feature of the invention. Examples of suchnucleic acids include those with one or a few silent or conservativenucleic acid substitutions as compared to a given nucleic acid sequence.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporated byreference.

The phrase “in solution” refers to an assay or reaction condition inwhich the components of the assay or reaction are not attached to asolid support. For example, certain assays of the invention includeincubating oligonucleotides together with HPV nucleic acids and HPVnucleic acid amplicons in solution to allow hybridization to occur.

The term “insertion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is added to the nucleicacid sequence, e.g., at a 5′-terminus, at a 3′-terminus, and/or at aninternal position of the nucleic acid sequence.

The “L1 region” refers to the L1 open reading frame of the humanpapillomavirus genome.

A “label” or “labeling moiety” refers to a moiety attached (covalentlyor non-covalently), or capable of being attached, to a molecule, whichmoiety provides or is capable of providing information about themolecule (e.g., descriptive, identifying, etc. information about themolecule) or another molecule with which the labeled molecule interacts(e.g., hybridizes, etc.). Exemplary labels include fluorescent labels(including, e.g., quenchers or absorbers), weakly fluorescent labels,non-fluorescent labels, colorimetric labels, chemiluminescent labels,bioluminescent labels, radioactive labels, mass-modifying groups,antibodies, antigens, biotin, haptens, enzymes (including, e.g.,peroxidase, phosphatase, etc.), and the like.

A “linker” refers to a chemical moiety that covalently or non-covalentlyattaches a compound or substituent group to another moiety, e.g., anucleic acid, an oligonucleotide probe, a primer nucleic acid, anamplicon, a solid support, or the like. For example, linkers areoptionally used to attach oligonucleotides to a solid support (e.g., ina linear or other logical oligonucleotide array). To further illustrate,a linker optionally attaches a label (e.g., a fluorescent dye, aradioisotope, etc.) to an oligonucleotide, a primer nucleic acid, or thelike. Linkers are typically at least bifunctional chemical moieties andin certain embodiments, they comprise cleavable attachments, which canbe cleaved by, e.g., heat, an enzyme, a chemical agent, electromagneticradiation, etc. to release materials or compounds from, e.g., a solidsupport. A careful choice of linker allows cleavage to be performedunder appropriate conditions compatible with the stability of thecompound and assay method. Generally a linker has no specific biologicalactivity other than to, e.g., join chemical species together or topreserve some minimum distance or other spatial relationship betweensuch species. However, the constituents of a linker may be selected toinfluence some property of the linked chemical species such asthree-dimensional conformation, net charge, hydrophobicity, etc.Exemplary linkers include, e.g., oligopeptides, oligonucleotides,oligopolyamides, oligoethyleneglycerols, oligoacrylamides, alkyl chains,or the like. Additional description of linker molecules is provided in,e.g., Hermanson, Bioconjugate Techniques, Elsevier Science (1996),Lyttle et al. (1996) Nucleic Acids Res. 24(14):2793, Shchepino et al.(2001) Nucleosides, Nucleotides, & Nucleic Acids 20:369, Doronina et al(2001) Nucleosides, Nucleotides, & Nucleic Acids 20:1007, Trawick et al.(2001) Bioconjugate Chem. 12:900, Olejnik et al. (1998) Methods inEnzymology 291:135, and Pljevaljcic et al. (2003) J. Am. Chem. Soc.125(12):3486, all of which are incorporated by reference.

A “mass modifying” group modifies the mass, typically measured in termsof molecular weight as daltons, of a molecule that comprises the group.

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction. An “amplificationreaction mixture” refers to a solution containing reagents necessary tocarry out an amplification reaction, and typically contains primers, athermostable DNA polymerase, dNTPs, and a divalent metal cation in asuitable buffer. A reaction mixture is referred to as complete if itcontains all reagents necessary to carry out the reaction, andincomplete if it contains only a subset of the necessary reagents. Itwill be understood by one of skill in the art that reaction componentsare routinely stored as separate solutions, each containing a subset ofthe total components, for reasons of convenience, storage stability, orto allow for application-dependent adjustment of the componentconcentrations, and that reaction components are combined prior to thereaction to create a complete reaction mixture. Furthermore, it will beunderstood by one of skill in the art that reaction components arepackaged separately for commercialization and that useful commercialkits may contain any subset of the reaction components which includesthe modified primers of the invention.

A “modified nucleotide substitution” in the context of anoligonucleotide refers to an alteration in which at least one nucleotideof the oligonucleotide sequence is replaced by a different nucleotidethat provides a desired property to the oligonucleotide. Exemplarymodified nucleotides that can be substituted in the oligonucleotidesdescribed herein include, e.g., a C5-methyl-dC, a C5-ethyl-dC, aC5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, aC5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, aC5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, aC7-propargylamino-dG, a 7-deaza-2-deoxyxanthosine, a pyrazolopyrimidineanalog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2′-0-methylRibo-U, 2′-0-methyl Ribo-C, an N4-ethyl-dC, an N6-methyl-dA, and thelike. Many other modified nucleotides that can be substituted in theoligonucleotides of the invention are referred to herein or areotherwise known in the art. In certain embodiments, modified nucleotidesubstitutions modify melting temperatures (T_(m)) of theoligonucleotides relative to the melting temperatures of correspondingunmodified oligonucleotides. To further illustrate, certain modifiednucleotide substitutions can reduce non-specific nucleic acidamplification (e.g., minimize primer dimer formation or the like),increase the yield of an intended target amplicon, and/or the like insome embodiments of the invention. Examples of these types of nucleicacid modifications are described in, e.g., U.S. Pat. No. 6,001,611,entitled “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS,” issued Dec. 14,1999 to Will, which is incorporated by reference.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, an oligonucleotideoptionally comprises a quencher moiety, a labeling moiety, or the like.

The term “nucleic acid” refers to nucleotides (e.g., ribonucleotides,deoxyribonucleotides, dideoxynucleotides, etc.) and polymers thatcomprise such nucleotides covalently linked together, either in a linearor branched fashion. Exemplary nucleic acids include deoxyribonucleicacids (DNAs), ribonucleic acids (RNAs), DNA-RNA hybrids,oligonucleotides, polynucleotides, genes, cDNAs, aptamers, antisensenucleic acids, molecular beacons, nucleic acid probes, peptide nucleicacids (PNAs), locked nucleic acids (LNAT™s), PNA-DNA conjugates, PNA-RNAconjugates, LNA™-DNA conjugates, LNA™-RNA conjugates, and the like.

A nucleic acid is typically single-stranded or double-stranded and willgenerally contain phosphodiester bonds, although in some cases, asoutlined herein, nucleic acid analogs are included that may havealternate backbones, including, for example and without limitation,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925 and thereferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al.(1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) ChemicaScripta 26:1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res.19:1437 and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.(1989) J. Am. Chem. Soc. 111:2321), O-methylphosphoroamidite linkages(Eckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press (1992)), and peptide nucleic acid backbones andlinkages (Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992)Chem. Int. Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566; andCarlsson et al. (1996) Nature 380:207), which references are eachincorporated by reference. Other analog nucleic acids include those withpositively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad.Sci. USA 92:6097); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghvi and P. Dan Cook,which references are each incorporated by reference. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (Jenkins et al. (1995) Chem. Soc. Rev. pp169-176, which is incorporated by reference). Several nucleic acidanalogs are also described in, e.g., Rawls, C & E News Jun. 2, 1997 page35, which is incorporated by reference. These modifications of theribose-phosphate backbone may be done to facilitate the addition ofadditional moieties such as labeling moieties, or to alter the stabilityand half-life of such molecules in physiological environments.

In addition to naturally occurring heterocyclic bases that are typicallyfound in nucleic acids (e.g., adenine, guanine, thymine, cytosine, anduracil); nucleic acid analogs also include those having non-naturallyoccurring heterocyclic or other modified bases, many of which aredescribed, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640,which are each incorporated by reference. To further illustrate, certainbases used in nucleotides that act as melting temperature (T_(m))modifiers are optionally included. For example, some of these include7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.),pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, entitled“SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINE NUCLEOTIDES,” which issued Nov.23, 1999 to Seela, which is incorporated by reference. Otherrepresentative heterocyclic bases include, e.g., hypoxanthine, inosine,xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine,2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine andxanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, and the like.

Additional examples of modified bases and nucleotides are also describedin, e.g., U.S. Pat. No. 5,484,908, entitled “OLIGONUCLEOTIDES CONTAINING5-PROPYNYL PYRIMIDINES,” issued Jan. 16, 1996 to Froehler et al., U.S.Pat. No. 5,645,985, entitled “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Jul.8, 1997 to Froehler et al., U.S. Pat. No. 5,830,653, entitled “METHODSOF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Nov. 3, 1998to Froehler et al., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al.,U.S. Pat. No. 6,303,315, entitled “ONE STEP SAMPLE PREPARATION ANDDETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct.16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905,entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al.that published May 15, 2003, which are each incorporated by reference.

A “nucleotide” refers to an ester of a nucleoside, e.g., a phosphateester of a nucleoside. To illustrate, a nucleotide can include 1, 2, 3,or more phosphate groups covalently linked to a 5′ position of a sugarmoiety of the nucleoside.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein- and/or nucleic acid-based catalyst that acts to reduce theactivation energy of a chemical reaction involving other compounds or“substrates.” A “nucleotide incorporating enzyme” refers to an enzymethat catalyzes the incorporation of nucleotides into a nucleic acid,e.g., during nucleic acid amplification or the like. Exemplarynucleotide incorporating enzymes include, e.g., polymerases, terminaltransferases, reverse transcriptases, telomerases, polynucleotidephosphorylases, and the like. A “thermostable enzyme” refers to anenzyme that is stable to heat, is heat resistant and retains sufficientcatalytic activity when subjected to elevated temperatures for selectedperiods of time. For example, a thermostable polymerase retainssufficient activity to effect subsequent primer extension reactions whensubjected to elevated temperatures for the time necessary to effectdenaturation of double-stranded nucleic acids. Heating conditionsnecessary for nucleic acid denaturation are well known in the art andare exemplified in U.S. Pat. Nos. 4,683,202 and 4,683,195, which areboth incorporated by reference. As used herein, a thermostablepolymerase is typically suitable for use in a temperature cyclingreaction such as the polymerase chain reaction (“PCR”). For athermostable polymerase, enzymatic activity refers to the catalysis ofthe combination of the nucleotides in the proper manner to form primerextension products that are complementary to a template nucleic acid(e.g., selected subsequences of an HPV L1 region).

A “nucleotide mismatch” in the context of an oligonucleotide and acorresponding target nucleic acid of the oligonucleotide refers to anucleotide difference or position of non-complementarity between theoligonucleotide and target nucleic acids when the two nucleic acids arealigned for maximum correspondence. For example, a cross-reactiveoligonucleotide of the invention may include one or more nucleotidemismatches with a substantially complementary sequence in the L1 regionof a given target high-risk HPV type.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22:1859-1862; the triester method of Matteucci et al. (1981) J.Am. Chem. Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, or other methods known in theart. All of these references are incorporated by reference.

The term “probe nucleic acid” or “probe” refers to a labeled orunlabeled oligonucleotide capable of selectively hybridizing to a targetnucleic acid under suitable conditions. Typically, a probe issufficiently complementary to a specific target sequence (e.g., the L1region of a high risk HPV type) contained in a nucleic acid sample toform a stable hybridization duplex with the target sequence under aselected hybridization condition, such as, but not limited to, astringent hybridization condition. A hybridization assay carried outusing the probe under sufficiently stringent hybridization conditionspermits the selective detection of a specific target sequence. The term“hybridizing region” refers to that region of a nucleic acid that isexactly or substantially complementary to, and therefore hybridizes to,the target sequence. For use in a hybridization assay for thediscrimination of single nucleotide differences in sequence, thehybridizing region is typically from about 8 to about 100 nucleotides inlength. Although the hybridizing region generally refers to the entireoligonucleotide, the probe may include additional nucleotide sequencesthat function, for example, as linker binding sites to provide a sitefor attaching the probe sequence to a solid support or the like. Incertain embodiments, a probe of the invention is included in a nucleicacid that comprises one or more labels (e.g., a reporter dye, a quenchermoiety, etc.), such as a 5′-nuclease probe, a FRET probe, a molecularbeacon, or the like, which can also be utilized to detect hybridizationbetween the probe and target nucleic acids in a sample. In someembodiments, the hybridizing region of the probe is completelycomplementary to the target sequence. However, in general, completecomplementarity is not necessary; stable duplexes may contain mismatchedbases or unmatched bases. Modification of the stringent conditions maybe necessary to permit a stable hybridization duplex with one or morebase pair mismatches or unmatched bases. Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001), which is incorporated byreference, provides guidance for suitable modification. Stability of thetarget/probe duplex depends on a number of variables including length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, temperature, and ionic conditions. One of skill in theart will recognize that, in general, the exact complement of a givenprobe is similarly useful as a probe. Exemplary probes of the invention,which bind to the L1 region of a high risk HPV type comprise sequencesselected from SEQ ID NOS: 1-31 and complements thereof. One of skill inthe art will also recognize that, in certain embodiments, probe nucleicacids can also be used as primer nucleic acids.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a template nucleic acid (e.g., the L1 region of a high risk HPV type)and permit chain extension or elongation using, e.g., a nucleotideincorporating biocatalyst, such as a polymerase under appropriatereaction conditions. A primer nucleic acid is typically a natural orsynthetic oligonucleotide (e.g., a single-strandedoligodeoxyribonucleotide, etc.). Although other primer nucleic acidlengths are optionally utilized, they typically comprise hybridizingregions that range from about 8 to about 100 nucleotides in length.Short primer nucleic acids generally utilize cooler temperatures to formsufficiently stable hybrid complexes with template HPV nucleic acids. Aprimer nucleic acid that is at least partially complementary to asubsequence of a template HPV nucleic acid is typically sufficient tohybridize with the template for extension to occur. A primer nucleicacid can be labeled, if desired, by incorporating a label detectable by,e.g., spectroscopic, photochemical, biochemical, immunochemical,chemical, or other techniques. To illustrate, useful labels includeradioisotopes, fluorescent dyes, electron-dense reagents, enzymes (ascommonly used in ELISAs), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available. Many of these and otherlabels are described further herein and/or otherwise known in the art.Exemplary primer nucleic acids of the invention, which bind to the L1region of a high risk HPV type comprise sequences selected from SEQ IDNOS: 1-31 and complements thereof. Other suitable primers are alsoreferred to herein and/or known in the art. One of skill in the art willrecognize that, in certain embodiments, primer nucleic acids can also beused as probe nucleic acids.

A “quencher moiety” or “quencher” refers to a moiety that reduces and/oris capable of reducing the detectable emission of radiation, e.g.,fluorescent or luminescent radiation, from a source that would otherwisehave emitted this radiation. A quencher typically reduces the detectableradiation emitted by the source by at least 50%, typically by at least80%, and more typically by at least 90%. Exemplary quenchers areprovided in, e.g., U.S. Pat. No. 6,465,175, entitled “OLIGONUCLEOTIDEPROBES BEARING QUENCHABLE FLUORESCENT LABELS, AND METHODS OF USETHEREOF,” which issued Oct. 15, 2002 to Horn et al., which isincorporated by reference.

The term “sample” refers to any substance containing or presumed tocontain HPV nucleic acid including, but not limited to, tissue or fluidisolated from one or more subjects or individuals, in vitro cell cultureconstituents, as well as clinical samples. Exemplary samples includeurine, seminal fluid, seminal plasma, prostatic fluid, vaginal fluid,cervical fluid, uterine fluid, cervical scrapings, amniotic fluid, analscrapings, mucus, sputum, tissue, and the like.

“Selectively hybridizing” or “selective hybridization” in the context ofnucleic acid hybridization refers to a nucleic acid that hybridizes orbinds to a target HPV nucleic acid (e.g., the L1 region of HPV types 33,35, 52, and/or 58) to a greater extent than the nucleic acid binds,under the same hybridization conditions, to non-target nucleic acids(e.g., the L1 region of HPV types 6, 11, 26, 40, 42, 43, 44, 53, 54, 55,57, 64, 66, 67, and 70).

A “set” refers to a collection of at least two molecule or sequencetypes, e.g., 4, 5, 10, 20, 50, 100, 1,000 or more molecule or sequencetypes. For example, certain aspects of the invention include reactionmixtures having sets of amplicons. A “subset” refers to any portion of aset.

A “sequence” of a nucleic acid refers to the order and identity ofnucleotides in the nucleic acid. A sequence is typically read in the 5′to 3′ direction.

A “solid support” refers to a solid material which can be derivatizedwith, or otherwise attached to, a chemical moiety, such as anoligonucleotide or the like. Exemplary solid supports include plates,microwell plates, beads, microbeads, fibers, whiskers, combs,hybridization chips, membranes, single crystals, ceramic layers,self-assembling monolayers, and the like.

An oligonucleotide is “specific” for a target sequence if the number ofmismatches present between the oligonucleotide and the target sequenceis less than the number of mismatches present between theoligonucleotide and non-target sequences that might be present in asample. Hybridization conditions can be chosen under which stableduplexes are formed only if the number of mismatches present is no morethan the number of mismatches present between the oligonucleotide andthe target sequence. Under such conditions, the target-specificoligonucleotide can form a stable duplex only with a target sequence.Thus, the use of target-specific primers under suitably stringentamplification conditions enables the specific amplification of thosesequences which contain the target primer binding sites. Similarly, theuse of target-specific oligonucleotides under suitably stringenthybridization conditions enables the detection of a specific targetsequence.

A “subsequence” or “segment” refers to any portion of an entire nucleicacid sequence.

A “substantially identical variant” in the context of nucleic acids,refers to two or more sequences that have at least 80%, typically atleast 85%, more typically at least 90%, and still more typically atleast 95% nucleotide or sequence identity to one another when comparedand aligned for maximum correspondence, as measured using, e.g., asequence comparison algorithm or by visual inspection. In certainembodiments, for example, a substantially identical variant of a nucleicacid includes one or more modified nucleotide substitutions relative toa corresponding unmodified nucleic acid. The substantial identitygenerally exists over a region of the sequences that is at least about15 nucleotides in length, more typically over a region that is at leastabout 20 nucleotides in length, and even more typically the sequencesare substantially identical over a region of at least about 25nucleotides in length. In some embodiments, for example, the sequencesare substantially identical over the entire length of the nucleic acidsbeing compared.

The term “substitution” in the context of a nucleic acid sequence refersto an alteration in which at least one nucleotide of the nucleic acidsequence is replaced by a different nucleotide.

The terms “target sequence,” “target region,” and “target nucleic acid”refer to a region of a nucleic acid which is to be amplified, detected,or otherwise analyzed.

II. Overview

The invention relates to the selective detection of high-risk HPV types,which are types that are commonly considered to be carcinogenic. Forexample, the cross-reactive oligonucleotides (e.g., probe nucleic acids,primer nucleic acids, etc.) described herein detectably bind, underselected assay conditions, to multiple high-risk HPV types, such asHPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58. In addition, certainoligonucleotides of the invention have sufficient specificity to excludethe detection of low-risk HPV types, thereby minimizing the occurrenceof false positives. Accordingly, high-risk HPV infections can be readilyand accurately diagnosed using the methods and reagents describedherein. Oligonucleotide specificity is further illustrated, for example,in the example provided below. Many other features of the invention arealso described herein.

To further illustrate, certain methods of the invention includeacquiring a sample from a patient and amplifying a target nucleic acidfrom the L1 region of HPV, if HPV is present in the sample. Essentiallyany nucleic acid amplification technique can be utilized or adapted foruse in amplifying the target HPV nucleic acid. In certain embodiments,for example, at least one version of a polymerase chain reaction is usedto generate multiple copies of the target nucleic acid. In someembodiments, labeled primer nucleic acids are used in theseamplification protocols to facilitate amplicon detection. Target HPVnucleic acids are typically amplified prior to or simultaneously (e.g.,for real-time detection) with being contacted with a cross-reactiveoligonucleotide of the invention. The oligonucleotide comprises anucleic acid with a sequence selected from SEQ ID NOS: 1-31, asubstantially identical variant thereof, or complements of SEQ ID NOS:1-31 and the variant. In some embodiments of the invention, bovine serumalbumin-conjugated oligonucleotides are attached to the surfaces ofmicrowell plate wells in which the target HPV nucleic acid is amplified.Optionally, the oligonucleotides of the invention are arrayed on othertypes of solid supports or are present in solution when contacted withthe target HPV nucleic acids and/or the amplicons of those targets.

These methods also generally include monitoring (e.g., at a single timepoint, at multiple discrete time points, continuously over a selectedtime period, etc.) binding between the amplicons and the cross-reactiveoligonucleotides to determine whether HPV is present in the particularsamples, e.g., to diagnose patients from which the samples were derived,to monitor courses' of treatment for patients diagnosed with HPVinfections, and/or the like. In certain embodiments, a detectedhigh-risk HPV is genotyped by determining the temperature (i.e., theT_(m)) at which a target HPV nucleic acid or amplicon thereofdissociates from a given oligonucleotide of the invention uponhybridization. These and many other approaches to detecting andgenotyping HPV using the cross-reactive oligonucleotides of theinvention are described further herein.

In addition to compositions and reaction mixtures, the invention alsorelates to kits and systems for detecting and genotyping high-risk HPV,and to related computers and computer readable media.

III. Cross-Reactive Oligonucleotides

The cross-reactive oligonucleotides (e.g., probe nucleic acids, primernucleic acids, etc.) of the invention bind to the L1 region of, e.g.,HPV31, HPV33, HPV35, HPV52, HPV56, and/or HPV58. HPV31 is furtherdescribed in, e.g., Hubert et al. (1999) “DNA replication of humanpapillomavirus type 31 is modulated by elements of the upstreamregulatory region that lie 5′ of the minimal origin,” J. Virol.73(3):1835-1845, which is incorporated by reference. HPV33 is alsodescribed in, e.g., Cole et al. (1986) “Genome organization andnucleotide sequence of human papillomavirus type 33, which is associatedwith cervical cancer” J. Virol. 58:991-995, which is incorporated byreference. HPV35 is further described in, e.g., Delius et al. (1994)“Primer-directed sequencing of human papillomavirus types” Curr. Top.Microbiol. Immunol. 186:13-31, which is incorporated by reference. HPV52is also described in, e.g., Delius et al. (1994), supra, and Shimoda etal. (1988) “Human papillomavirus type 52: a new virus associated withcervical neoplasia” J Gen Virol. 69(11):2925-8, which are bothincorporated by reference. HPV56 is also described in, e.g., Delius etal. (1994) “Primer-directed sequencing of human papillomavirus types”Curr. Top. Microbiol. Immunol. 186:13-31, which is incorporated byreference. HPV58 is further described in, e.g., Kirii et al. (1991)“Human papillomavirus type 58 DNA sequence” Virology 185:424-427, whichis incorporated by reference. See also, the HPV Sequence Databaseprovided on the world wide web at hpv-web.lanl.gov as of May 7, 2004.

More specifically, the oligonucleotides of the invention each include anucleic acid with a sequence selected from SEQ ID NOS: 1-31, asubstantially identical variant thereof in which the variant has atleast 80% sequence identity to one of SEQ ID NOS: 1-31, and complementsof SEQ ID NOS: 1-31 and the variant. SEQ ID NOS: 1-31 are shown in TableI.

TABLE I SEQ ID NO SEQUENCE DESIGNATION 1 5′-CAGTACTAAAAGTCATGTTAGTGCT-3′A5256A 2 5′-CAGTACAAATAGTCATGTTAGTGCT-3′ A5256B 35′-TCATTTTTATATGTGCTTTCCTT-3′ A5258A 4 5′-ATTATCATTTTTATATGTACTTTCCTT-3′A5258B 5 5′-TCATTTTTATATGTGCCTTCCTT-3′ A5258C 65′-TTAAAATTTTCATTTTTATATGTACTTTCCTT-3′ A5258D 75′-CAGTACATAAAGTCATGTTAGTGCT-3′ A5256C 8 5′-TTAAAATTTTCATTTTTATATGT-3′A3352A 9 5′-CATAAAGTCATGTTAGTGCTGCGAGT-3′ A3352B 105′-ATTTTTATATGTGCTTTCCTTTTAATTGCAGCACAAACAGACA-3′ A3152HYB 115′-AATTGCAGCACAAACAGACAATTTTTATATGTGCTTTCCTTTT-3′ A5231HYB 125′-TTAAAATTTTCATTTTTATATGTACTTTC-3′ A3X5XA 135′-AAATTTTCATTTTTATATGTACTTTC-3′ A3X5XB 145′-TTAAAATTGTCATTTTTATATGTACTTTC-3′ A3X5XE 155′-TTAAAATPTPCATTTTTATATGPAETTPC-3′ A3X5XC 165′-TTAAAATZTZCATTTTTATATGZAJTTZC-3′ A3X5XD 175′-TTAAAAPPTPEAPPPPPAPAPGPAETTPC-3′ A3X5XH 185′-CATAAAGTCATATTAGTGCTGCGAGTGGTATC-3′ A3352C 195′-CATAAAGTCAPATTAGTGETGEGAGTGGTATC-3′ A3352D 205′-TTAAAATPGPCATTTTTATATGPAETTPC-3′ A3X5XF 215′-TTAAAAPPGPEAPPPPPAPAPGPAETTPC-3′ A3X5XG 225′-TATTCEPPAAAAPPGPEAPTTTTAPAPGPAEPPPE-3′ A3X5XY5S 235′-FTATTCQEPPAAAAPPGPEAPTTTTAPAPGPAEPPPEO-3′ FQIA3X5XY5S 245′-FTATTQCCTTAAAATPTPCATTTTTATATGPAETTPCO-3′ FQIA3X5XA4 255′-FTAPPQECPPAAAAPPTPEAPTTTTATAPGPAETTPEO-3′ FQA3X5XA15S 265′-FTATTQCCTTAAAATPGPCATTTTTATATGPGETGPCO-3′ FQIA3X5XZ4 275′-FTAPPQECPPAAAAPPGPEAPTTTTATAPGPGETGPEO-3′ FQA3X5XZ15S 285′-FTAPPQCCTTAAAATPGPCATTTTTATATGPGETGPCO-3′ FQIA3X5XS4 295′-FTATTQCCTTAAAATPGPCATTTTTATATGPAEPTPCO-3′ FQIA3X5XY4 305′-FTATTQCEPPAAAAPPGPEATTTTTATAPGPAEPTPCO-3′ FQIA3X5XY12S 315′-FTATTCQEPPAAAAPPGPEAPTTTTAPAPGPAEPPPEO-3′ FQIA3X5XY5As shown in Table I, P represents 5-propynyl-dU, E represents5-methyl-dC, Z represents 2′-O-methyl Ribo-U, F represents FAM, Qrepresents Black Hole Quencher™-2 (BHQ2), O represents phosphate, and Jrepresents 2′-0-methyl Ribo-C. To illustrate, oligonucleotide A3X5XA(SEQ ID NO: 12) has a sequence that has a single mismatch with HPV33 anda single mismatch with HPV52. In addition, oligonucleotide A3X5XA hastwo mismatches with HPV35 and two mismatches with HPV58. Certainperformance characteristics of oligonucleotide A3X5XA and otheroligonucleotides are provided below in the example.

The introduction of modified nucleotide substitutions intooligonucleotide sequences can, e.g., increase the melting temperature ofthe oligonucleotides. In certain embodiments, this can yield greatersensitivity relative to corresponding unmodified oligonucleotides evenin the presence of one or more mismatches in sequence between the targetnucleic acid and the cross-reactive oligonucleotide. To furtherillustrate, oligonucleotide A3X5XC (SEQ ID NO: 15) includes fivemodified nucleotide substitutions (5-propynyl dU and 5-methyl dC). Thesesubstitutions are placed near positions of mismatch with the targetnucleic acid sequence, e.g., in order to offset the destabilizing effectof a mismatch on the oligonucleotide:target hybrid. OligonucleotideA3X5XC is also described in the example provided below.

Additional variants of SEQ ID NOS: 1-31, which include otheralterations, are also optionally utilized. For example, variants of SEQID NOS: 1-31 can include one or more insertions, deletions, orsubstitutions relative to oligonucleotides that comprise nucleic acidswith sequences selected from SEQ ID NOS: 1-31. Other exemplary modifiednucleotides that can be substituted in the oligonucleotides of theinvention include, e.g., C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU,2,6-diaminopurines, C5-propynyl-dC, C7-propynyl-dA, C7-propynyl-dG,C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA,C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidineanalogs, pseudo-dU, nitro pyrrole, nitro indole, 2′-0-methyl Ribo-U,2′-0-methyl Ribo-C, an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a7-deaza-dG, N4-ethyl-dC, N6-methyl-dA, etc. To further illustrate, otherexamples of modified oligonucleotides include those having one or moreLNA™ monomers. Nucleotide analogs such as these are described furtherin, e.g., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF [2.2.1]BICYCLONUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al., U.S. Pat. No.6,303,315, entitled “ONE STEP SAMPLE PREPARATION AND DETECTION OFNUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct. 16, 2001 toSkouv, and U.S. Pat. Application Pub. No. 2003/0092905, entitled“SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al. thatpublished May 15, 2003, which are each incorporated by reference.Oligonucleotides comprising LNA™ monomers are available through, e.g.,Exiqon AIS (Vedbæk, D K). Additional oligonucleotide modifications arereferred to herein, including in the definitions provided above. It willbe appreciated that many of these modifications are also optionallyincorporated into primer nucleic acids used in performing the methods ofthe present invention. Other aspects of the probes and primers utilizedas described herein, including synthesis and labeling, are providedbelow.

Although other lengths are optionally utilized, the oligonucleotides ofthe invention generally comprise sequences that are typically betweenabout 12 and about 100 nucleotides in length, more typically betweenabout 15 and about 75 nucleotides in length, still more typicallybetween about 20 and about 50 nucleotides in length, and even moretypically between about 23 and about 35 nucleotides in length (e.g.,about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 nucleotides inlength). Methods of preparing oligonucleotides, such as nucleic acidsynthesis, are described further below.

Various approaches can be utilized by one of skill in the art to designoligonucleotides (e.g., substantially identical variants of nucleicacids having sequences selected from SEQ ID NOS: 1-31 or complementsthereof) that selectively hybridize to the L1 region of HPV types 33,35, 52, and/or 58. To illustrate, the DNAstar software package availablefrom DNASTAR, Inc. (Madison, Wis.) can be used for sequence alignments.For example, nucleic acid sequences from the L1 region of HPV types 33,35, 52, and/or 58 and non-target sequences can be uploaded into DNAstarEditSeq program as individual files. To further illustrate, pairs ofsequence files can be opened in the DNAstar MegAlign sequence alignmentprogram and the Clustal W method of alignment can be applied. Theparameters used for Clustal W alignments are optionally the defaultsettings in the software. MegAlign typically does not provide a summaryof the percent identity between two sequences. This is generallycalculated manually. From the alignments, regions having, e.g., lessthan 85% identity with one another are typically identified andoligonucleotide sequences in these regions can be selected. Many othersequence alignment algorithms and software packages are also optionallyutilized. Sequence alignment algorithms are also described in, e.g.,Mount, Bioinformatics: Sequence and Genome Analysis, Cold Spring HarborLaboratory Press (2001), and Durbin et al., Biological SequenceAnalysis: Probabilistic Models of Proteins and Nucleic Acids, CambridgeUniversity Press (1998), which are both incorporated by reference.

To further illustrate, optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman(1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA85:2444, which are each incorporated by reference, and by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group(Madison, Wis.), or by even by visual inspection.

Another example algorithm that is suitable for determining percentsequence identity is the BLAST algorithm, which is described in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410, which is incorporatedby reference. Software for performing versions of BLAST analyses ispublicly available through the National Center for BiotechnologyInformation on the world wide web at ncbi.nlm.nih.gov/ as of May 7,2004. This algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W in the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as the neighborhood word score threshold(Altschul et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915, which is incorporated by reference).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787, which is incorporated by reference). One measureof similarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a nucleic acid is considered similar to areference sequence (and, therefore, homologous) if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, or less than about 0.01, and oreven less than about 0.001.

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J. Mol. Evol. 35:351-360, which isincorporated by reference. The method used is similar to the methoddescribed by Higgins & Sharp (1989) CABIOS 5:151-153, which isincorporated by reference. The program can align, e.g., up to 300sequences of a maximum length of 5,000 letters. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This clustercan then be aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences can be aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program can also be used to plot a dendogram or treerepresentation of clustering relationships. The program is run bydesignating specific sequences and their amino acid or nucleotidecoordinates for regions of sequence comparison.

In practicing the present invention, many conventional techniques inmolecular biology and recombinant DNA are optionally used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger), DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D.N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal,1984, A Practical Guide to Molecular Cloning; the series, Methods inEnzymology (Academic Press, Inc.); Gene Transfer Vectors for MammalianCells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory); Methods in Enzymology Vol. 154 and Vol. 155 (Wu andGrossman, and Wu, eds., respectively), all of which are incorporated byreference.

IV. Sequence Variations

Numerous nucleic acid and polypeptide sequences are within the scope ofthe present invention, whether as target sequences or the agents used todetect those target sequences.

Silent Variations

It will be appreciated by those skilled in the art that due to thedegeneracy of the genetic code, a multitude of nucleic acids sequencesfrom the L1 open reading frame of high-risk HPV types (e.g., HPV31,HPV33, HPV35, HPV52, HPV56, and/or HPV58) may be produced, some of whichmay bear minimal sequence homology to the nucleic acid sequencesexplicitly disclosed herein. For instance, inspection of the codon table(Table II) shows that codons AGA, AGG, CGA, CGC, CGG, and CGU all encodethe amino acid arginine. Thus, at every position in the nucleic acids ofthe invention where an arginine is specified by a codon, the codon canbe altered to any of the corresponding codons described above withoutaltering an encoded polypeptide. It is understood that U in an RNAsequence corresponds to T in a DNA sequence.

TABLE II Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Such “silent variations” are one species of “conservatively modifiedvariations”, discussed below. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid, which encodes a polypeptide is implicit in anydescribed sequence. The invention provides each and every possiblevariation of nucleic acid sequence of the L1 open reading frame ofHPV31, HPV33, HPV35, HPV52, HPV56, and HPV58 that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode (e.g., as set forth in Table II) as applied to the nucleic acidsequences of these open reading frames. All such variations of everynucleic acid herein are specifically provided and described byconsideration of the sequence in combination with the genetic code.

Conservative Variations

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids, which encode identical or essentially identical aminoacid sequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than 5%, more typically less than 4%, 2% or 1%) inan encoded sequence are “conservatively modified variations” where thealterations result in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table III sets forth six groups, whichcontain amino acids that are “conservative substitutions” for oneanother.

TABLE III Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Thus, “conservatively substituted variations” of a polypeptide encodedby the L1 open reading frame of HPV31, HPV33, HPV35, HPV52, HPV 56, andHPV58 referred to herein include substitutions of a small percentage,typically less than 5%, more typically less than 2% or 1%, of the aminoacids of the polypeptide sequence, with a conservatively selected aminoacid of the same conservative substitution group.

The addition of sequences that do not alter the encoded activity of anucleic acid molecule, such as the addition of a non-functionalsequence, is a conservative variation of the basic nucleic acid.

One of skill will appreciate that many conservative variations of thenucleic acids described herein yield a functionally identical nucleicacid. For example, as discussed above, owing to the degeneracy of thegenetic code, “silent substitutions” (i.e., substitutions in a nucleicacid sequence which do not result in an alteration in an encodedpolypeptide) are an implied feature of every nucleic acid sequence,which encodes an amino acid. Similarly, “conservative amino acidsubstitutions,” in one or a few amino acids in an amino acid sequenceare substituted with different amino acids with highly similarproperties, are also readily identified as being highly similar to adisclosed construct. Such conservative variations of each disclosedsequence are a feature of the present invention.

V. Probe and Primer Preparation

Many different primer pairs are optionally used to amplify target HPVDNA. Examples of suitable primer pairs directed to conserved regionsamong different HPV types in the L1 region include MY11/MY09 andGP5/GP6. These primers are also described in, e.g., Manos et al. (1989)“The use of polymerase chain reaction amplification for the detection ofgenital human papillomaviruses” Cancer Cells 7:209-214, and Van denBrule et al. (1990) “General primer-mediated polymerase chain reactionpermits the detection of sequenced and still unsequenced humanpapillomavirus genotypes in cervical scrapes and carcinomas” Int. J.Cancer 45:644-649, which are both incorporated by reference. These andother primer pairs are also described in, e.g., U.S. Pat. Nos. 6,482,588and 5,705,627, U.S. Pat. Application Pub. No. US 2003/0059806 A1, andEuropean Pat. Application Pub. No. EP 1302550 A1, which are eachincorporated by reference.

The oligonucleotide probes and primers of the invention are optionallyprepared using essentially any technique known in the art. In certainembodiments, for example, the oligonucleotide probes and primersdescribed herein are synthesized chemically using essentially anynucleic acid synthesis method, including, e.g., according to the solidphase phosphoramidite method described by Beaucage and Caruthers (1981),Tetrahedron Letts. 22(20):1859-1862, which is incorporated by reference.To further illustrate, oligonucleotides can also be synthesized using atriester method (see, e.g., Capaldi et al. (2000) “Highly efficientsolid phase synthesis of oligonucleotide analogs containingphosphorodithioate linkages” Nucleic Acids Res. 28(9):e40 and Eldrup etal. (1994) “Preparation of oligodeoxyribonucleoside phosphorodithioatesby a triester method” Nucleic Acids Res. 22(10):1797-1804, which areboth incorporated by reference). Other synthesis techniques known in theart can also be utilized, including, e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al. (1984) NucleicAcids Res. 12:6159-6168, which is incorporated by reference. A widevariety of equipment is commercially available for automatedoligonucleotide synthesis. Multi-nucleotide synthesis approaches (e.g.,tri-nucleotide synthesis, etc.) are also optionally utilized. Moreover,the primer nucleic acids described herein optionally include variousmodifications. In certain embodiments, for example, primers includerestriction site linkers, e.g., to facilitate subsequent ampliconcloning or the like. To further illustrate, primers are also optionallymodified to improve the specificity of amplification reactions asdescribed in, e.g., U.S. Pat. No. 6,001,611, entitled “MODIFIED NUCLEICACID AMPLIFICATION PRIMERS,” issued Dec. 14, 1999 to Will, which isincorporated by reference. Primers and probes can also be synthesizedwith various other modifications as described herein or as otherwiseknown in the art.

Essentially any label is optionally utilized to label the probes and/orprimers described herein. In some embodiments, for example, the labelcomprises a fluorescent dye (e.g., a rhodamine dye (e.g., R6G, R110,TAMRA, ROX, etc.), a fluorescein dye (e.g., JOE, VIC, TET, HEX, FAM,etc.), a halofluorescein dye, a cyanine dye (e.g., CY3, CY3.5, CY5,CY5.5, etc.), a BODIPY® dye (e.g., FL, 530/550, TR, TMR, etc.), an ALEXAFLUOR® dye (e.g., 488, 532, 546, 568, 594, 555, 653, 647, 660, 680,etc.), a dichlororhodamine dye, an energy transfer dye (e.g., BIGDYE™ v1 dyes, BIGDYE™ v 2 dyes, BIGDYE™ v 3 dyes, etc.), Lucifer dyes (e.g.,Lucifer yellow, etc.), CASCADE BLUE®, Oregon Green, and the like.Additional examples of fluorescent dyes are provided in, e.g., Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Products,Ninth Ed. (2003) and the updates thereto, which are each incorporated byreference. Fluorescent dyes are generally readily available from variouscommercial suppliers including, e.g., Molecular Probes, Inc. (Eugene,Oreg.), Amersham Biosciences Corp. (Piscataway, N.J.), AppliedBiosystems (Foster City, Calif.), etc. Other labels include, e.g.,biotin, weakly fluorescent labels (Yin et al. (2003) Appl EnvironMicrobiol. 69(7):3938, Babendure et al. (2003) Anal. Biochem. 317(1):1,and Jankowiak et al. (2003) Chem Res Toxicol. 16(3):304),non-fluorescent labels, colorimetric labels, chemiluminescent labels(Wilson et al. (2003) Analyst. 128(5):480 and Roda et al. (2003)Luminescence 18(2):72), Raman labels, electrochemical labels,bioluminescent labels (Kitayama et al. (2003) Photochem Photobiol.77(3):333, Arakawa et al. (2003) Anal. Biochem. 314(2):206, and Maeda(2003) J. Pharm. Biomed. Anal. 30(6):1725), and an alpha-methyl-PEGlabeling reagent as described in, e.g., U.S. Provisional PatentApplication No. 60/428,484, filed on Nov. 22, 2002, which references areeach incorporated by reference. Nucleic acid labeling is also describedfurther below.

In addition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asThe Midland Certified Reagent Company, The Great American Gene Company,ExpressGen Inc., Operon Technologies Inc., Proligo LLC, and many others.

VI. Sample Preparation and Nucleic Acid Amplification

Samples are generally derived or isolated from subjects, typicallymammalian subjects, more typically human subjects, e.g., suspected ofhaving HPV infections or as part of screening examinations. Exemplarysamples or specimens include urine, seminal fluid, seminal plasma,prostatic fluid, vaginal fluid, cervical fluid, uterine fluid, cervicalscrapings, amniotic fluid, anal scrapings, mucus, sputum, tissue, andthe like. Essentially any technique for acquiring these samples isoptionally utilized including, e.g., scraping, venipuncture, swabbing(e.g., using a cervical swab or brush), biopsy, or other techniquesknown in the art. Methods of storing specimens, culturing cells,extracting or otherwise isolating and preparing nucleic acids from thesesources are generally known in the art and many of these are describedfurther in the references provided herein. For example, one of the mostpowerful and basic technologies for deriving and detecting nucleic acidsis nucleic acid amplification. In the present invention, amplificationof nucleic acids of interest typically precedes or is concurrent withthe detection of that DNA. In addition, the oligonucleotides describedherein are also optionally amplified, e.g., following chemical synthesisor the like. In some embodiments, detectable signals are amplified,e.g., using branched nucleic acid or other signal amplification formatsknown in the art.

Amplification methods that are optionally utilized include, e.g.,various polymerase or ligase mediated amplification methods, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), and/orthe like. Details regarding the use of these and other amplificationmethods can be found in any of a variety of standard texts, including,e.g., Berger, Sambrook, Ausubel, and PCR Protocols A Guide to Methodsand Applications (Innis et al. eds) Academic Press, Inc., San Diego,Calif. (1990) (Innis), all of which are incorporated by reference. Manyavailable biology texts also have extended discussions regarding PCR andrelated amplification methods. Nucleic acid amplification is alsodescribed in, e.g., Mullis et al., (1987) U.S. Pat. No. 4,683,202 andSooknanan (1995) Biotechnology 13:563, which are both incorporated byreference. Improved methods of amplifying large nucleic acids by PCR aresummarized in Cheng et al. (1994) Nature 369:684, which is incorporatedby reference. In certain embodiments, duplex PCR is utilized to amplifytarget nucleic acids. Duplex PCR amplification is described further in,e.g., Gabriel et al. (2003) “Identification of human remains byimmobilized sequence-specific oligonucleotide probe analysis of mtDNAhypervariable regions I and II,” Croat. Med. J. 44(3) 293 and La et al.(2003) “Development of a duplex PCR assay for detection of Brachyspirahyodysenteriae and Brachyspira pilosicoli in pig feces,” J. Clin.Microbiol. 41(7):3372, which are both incorporated by reference.Optionally, labeled primers (e.g., biotinylated primers, etc.) areutilized to amplify nucleic acids in a sample, e.g., to facilitatedetection of hybridization between amplicons and the oligonucleotides ofthe invention. Labeling is described further herein.

Amplicons are optionally recovered and purified from other reactioncomponents by any of a number of methods well known in the art,including electrophoresis, chromatography, precipitation, dialysis,filtration, and/or centrifugation. Aspects of nucleic acid purificationare described in, e.g., Douglas et al., DNA Chromatography, Wiley, John& Sons, Inc. (2002), and Schott, Affinity Chromatography: TemplateChromatography of Nucleic Acids and Proteins, Chromatographic ScienceSeries, #27, Marcel Dekker (1984), all of which are incorporated byreference. In certain embodiments, amplicons are not purified prior todetection. The detection of amplicons is described further below.

VII. Oligonucleotide Arrays

In certain embodiments of the invention, the oligonucleotides describedherein are covalently or noncovalently attached to solid supports whichare then contacted with samples comprising amplified and labeled nucleicacid from a subject. In other embodiments, the oligonucleotides of theinvention are provided free in solution. Essentially any substratematerial is optionally adapted for use in these aspects of theinvention. In certain embodiments, for example, substrates arefabricated from silicon, glass, or polymeric materials (e.g., glass orpolymeric microscope slides, silicon wafers, etc.). Suitable glass orpolymeric substrates, including microscope slides, are available fromvarious commercial suppliers, such as Fisher Scientific (Pittsburgh,Pa.) or the like. In some embodiments, solid supports utilized in theinvention are membranes. Suitable membrane materials are optionallyselected from, e.g. polyaramide membranes, polycarbonate membranes,porous plastic matrix membranes (e.g., POREX® Porous Plastic, etc.),porous metal matrix membranes, polyethylene membranes, poly(vinylidenedifluoride) membranes, polyamide membranes, nylon membranes, ceramicmembranes, polyester membranes, polytetrafluoroethylene (TEFLON®)membranes, woven mesh membranes, microfiltration membranes,nanofiltration membranes, ultrafiltration membranes, dialysis membranes,composite membranes, hydrophilic membranes, hydrophobic membranes,polymer-based membranes, a non-polymer-based membranes, powderedactivated carbon membranes, polypropylene membranes, glass fibermembranes, glass membranes, nitrocellulose membranes, cellulosemembranes, cellulose nitrate membranes, cellulose acetate membranes,polysulfone membranes, polyethersulfone membranes, polyolefin membranes,or the like. Many of these membranous materials are widely availablefrom various commercial suppliers, such as, P. J. Cobert Associates,Inc. (St. Louis, Mo.), Millipore Corporation (Bedford, Mass.), or thelike. Other exemplary solid supports that are optionally utilizedinclude, e.g., ceramics, metals, resins, gels, plates, microwell plates,beads, microbeads, whiskers, fibers, combs, single crystals, andself-assembling monolayers.

The oligonucleotides of the invention are directly or indirectly (e.g.,via linkers, such as bovine serum albumin (BSA) or the like) attached tothe supports, e.g., by any available chemical or physical method. A widevariety of linking chemistries are available for linking molecules to awide variety of solid supports. More specifically, nucleic acids may beattached to the solid support by covalent binding such as by conjugationwith a coupling agent or by non-covalent binding such as electrostaticinteractions, hydrogen bonds or antibody-antigen coupling, or bycombinations thereof. Typical coupling agents include biotin/avidin,biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F_(c)fragment, and streptavidin/protein A chimeras (Sano et al. (1991)Bio/Technology 9:1378, which is incorporated by reference), orderivatives or combinations of these agents. Nucleic acids may beattached to the solid support by a photocleavable bond, an electrostaticbond, a disulfide bond, a peptide bond, a diester bond or a combinationof these bonds. Nucleic acids are also optionally attached to solidsupports by a selectively releasable bond such as 4,4′-dimethoxytritylor its derivative. Derivatives which have been found to be usefulinclude 3 or 4[bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4[bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4[bis-(4-methoxyphenyl)]-hydroxymethyl-benzoic acid,N-succinimidyl-3 or 4[bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid,and salts of these acids.

As referred to above, oligonucleotides are optionally attached to solidsupports via linkers between the nucleic acids and the solid support.Useful linkers include a coupling agent, as described above for bindingto other or additional coupling partners, or to render the attachment tothe solid support cleavable.

Cleavable attachments can be created by attaching cleavable chemicalmoieties between the oligonucleotides and the solid support including,e.g., an oligopeptide, oligonucleotide, oligopolyamide, oligoacrylamide,oligoethylene glycerol, alkyl chains of between about 6 to 20 carbonatoms, and combinations thereof. These moieties may be cleaved with,e.g., added chemical agents, electromagnetic radiation, or enzymes.Exemplary attachments cleavable by enzymes include peptide bonds whichcan be cleaved by proteases, and phosphodiester bonds which can becleaved by nucleases.

Chemical agents such as β-mercaptoethanol, dithiothreitol (DTT) andother reducing agents cleave disulfide bonds. Other agents which may beuseful include oxidizing agents, hydrating agents and other selectivelyactive compounds. Electromagnetic radiation such as ultraviolet,infrared and visible light cleave photocleavable bonds. Attachments mayalso be reversible, e.g., using heat or enzymatic treatment, orreversible chemical or magnetic attachments. Release and reattachmentcan be performed using, e.g., magnetic or electrical fields.

Array based hybridization is particularly suitable for detecting HPVnucleic acids, as it can be used to detect the presence of manyamplicons simultaneously. A number of array systems have been describedand can be adapted for use with the present invention. Aspects of arrayconstruction and use are also described in, e.g., Sapolsky et al. (1999)“High-throughput polymorphism screening and genotyping with high-densityoligonucleotide arrays.” Genetic Analysis Biomolecular Engineering14:187-192; Lockhart (1998) “Mutant yeast on drugs” Nature Medicine4:1235-1236; Fodor (1997) “Genes, Chips and the Human Genome.” FASEBJournal 11:A879; Fodor (1997) “Massively Parallel Genomics” Science 277:393-395; and Chee et al. (1996) “Accessing Genetic Information withHigh-Density DNA Arrays” Science 274:610-614, all of which areincorporated by reference.

Other probes and primers for detecting HPV nucleic acids, which areoptionally utilized in addition to the probes and primer described aboveto perform the methods and other aspects of the invention, are describedin, e.g., U.S. Pat. No. 5,705,627 to Manos et al., which is incorporatedby reference.

VIII. Nucleic Acid Hybridization

Hybridization of oligonucleotides to their target HPV nucleic acids canbe accomplished by choosing the appropriate hybridization conditions.The stability of the oligonucleotide:target nucleic acid hybrid istypically selected to be compatible with the assay and washingconditions so that stable, detectable hybrids form only between theoligonucleotides and target HPV nucleic acids. Manipulation of one ormore of the different assay parameters determines the exact sensitivityand specificity of a particular hybridization assay.

More specifically, hybridization between complementary bases of DNA,RNA, PNA, or combinations of DNA, RNA and PNA, occurs under a widevariety of conditions that vary in temperature, salt concentration,electrostatic strength, buffer composition, and the like. Examples ofthese conditions and methods for applying them are described in, e.g.,Tijssen (1993), supra, and Hames and Higgins, supra. Hybridizationgenerally takes place between about 0° C. and about 70° C., for periodsof from about one minute to about one hour, depending on the nature ofthe sequence to be hybridized and its length. However, it is recognizedthat hybridizations can occur in seconds or hours, depending on theconditions of the reaction. To illustrate, typical hybridizationconditions for a mixture of two 20-mers is to bring the mixture to 68°C., followed by cooling to room temperature (22° C.) for five minutes orat very low temperatures such as 2° C. in 2 microliters. Hybridizationbetween nucleic acids may be facilitated using buffers such as Tris-EDTA(TE), Tris-HCl and HEPES, salt solutions (e.g. NaCl, KCl, CaCl₂), orother aqueous solutions, reagents and chemicals. Examples of thesereagents include single-stranded binding proteins such as Rec A protein,T4 gene 32 protein, E. coli single-stranded binding protein and major orminor nucleic acid groove binding proteins. Other examples of suchreagents and chemicals include divalent ions, polyvalent ions andintercalating substances such as ethidium bromide, actinomycin D,psoralen, and angelicin. An exemplary hybridization procedure of use inthe present invention follows similar conditions as specified in theAMPLICOR® HPV Test protocol (Roche Diagnostics Corporation,Indianapolis, Ind.).

IX. Detection and Oligonucleotide Variations

As referred to above, amplified target HPV nucleic acid in the samplesutilized in the methods of the invention is optionally labeled to permitdetection of oligonucleotide-target hybridization duplexes. In general,a label can be any moiety which can be attached, e.g., to a primerutilized for amplification and provide a detectable signal (e.g., aquantifiable signal). Labels may be attached to a primer directly orindirectly by a variety of techniques known in the art. Depending on thetype of label used, the label can be attached to a terminal (5′ or 3′end of the primer) or a non-terminal nucleotide, and can be attachedindirectly through linkers or spacer arms of various sizes andcompositions. Using commercially available phosphoramidite reagents, onecan produce oligomers containing functional groups (e.g., thiols orprimary amines) at either the 5′ or 3′ terminus via an appropriatelyprotected phosphoramidite, and can label such oligonucleotides usingprotocols described in, for example, PCR Protocols: A Guide to Methodsand Applications (Innis et al, eds. Academic Press, Inc. (1990)), whichis incorporated by reference. In one embodiment, the label consists of abiotin molecule covalently bound to the primer at the 5′ end. The term“biotinylated primer” refers to a primer with one or more biotinmolecules bound either directly to the primer or indirectly throughintervening linker molecules.

To further illustrate, detection of oligonucleotide-target hybridizationduplexes is optionally by a chemiluminescent assay using a luminol-basedreagent as described in, e.g., Whitehead, et al. (1983) Nature30(5):158, which is incorporated by reference, and availablecommercially. Following hybridization of the oligonucleotide with thelabeled target DNA, the biotin molecule attached to the target DNA isconjugated, e.g., to streptavidin-horseradish peroxidase (SA-HRP).Alternatively, the target DNA can be labeled with horseradish peroxidasedirectly, thereby eliminating the separate conjugation step. In eithercase, subsequent oxidation of luminol by the horseradish peroxidaseenzyme results in the emission of photons, which is then detected, e.g.,on standard autoradiography film. The intensity of the signal is afunction of DNA quantity. A series of DNA standards containing knownamounts of DNA are typically assayed along with one or more unknownsamples. The signal intensities of the known DNA standards allows anempirical determination of the functional relationship between signalintensity and DNA quantity, which enables the quantitation of theunknown samples. Many other methods of detection are also optionallyutilized to perform the methods of the invention and are referred to inthe references cited herein and/or generally known in the art.

Any available method for detecting HPV amplicons can be used in thepresent invention. Common approaches include real time amplificationdetection with molecular beacons or 5′-nuclease probes, detection ofintercalating dyes, detection of labels incorporated into theamplification probes or the amplified nucleic acids themselves (e.g.,following electrophoretic separation of the amplification products fromunincorporated label), hybridization based assays (e.g., array basedassays) and/or detection of secondary reagents that bind to the nucleicacids. For example, a molecular beacon or a 5′-nuclease probe isoptionally designed to include an oligonucleotide of the invention(i.e., is selected from SEQ ID NOS: 1-31) or complements thereto), whichmolecular beacon or 5′-nuclease probe can be used to detect HPVamplicons. Molecular beacons or 5′-nuclease probes are described furtherbelow. Details on these general approaches are found in the referencescited herein, e.g., Sambrook and Ausubel. Additional strategies forlabeling nucleic acids and corresponding detection strategies can befound, e.g., in Haugland (2003) Handbook of Fluorescent Probes andResearch Chemicals Ninth Edition by Molecular Probes, Inc. (Eugene,Oreg.), which is incorporated by reference.

Molecular beacons (MBs) are oligonucleotides designed for real timedetection and quantification of target nucleic acids (e.g., target HPVamplicons). The 5′ and 3′ termini of MBs collectively comprise a pair ofmoieties which confers the detectable properties of the MB. One of thetermini is attached to a fluorophore and the other is attached to aquencher molecule capable of quenching a fluorescent emission of thefluorophore. For example, one example fluorophore-quencher pair can usea fluorophore such as EDANS or fluorescein, e.g., on the 5′-end and aquencher such as Dabcyl, e.g., on the 3′-end. When the MB is presentfree in solution, i.e., not hybridized to a second nucleic acid, thestem of the MB is stabilized by complementary base pairing. Thisself-complementary pairing results in a “hairpin loop” structure for theMB in which the fluorophore and the quenching moieties are proximal toone another. In this confirmation, the fluorescent moiety is quenched bythe fluorophore. The loop of the molecular beacon typically comprises anoligonucleotide described herein (e.g., an oligonucleotide selected fromSEQ ID NOS: 1-31 or complements thereto) and is accordinglycomplementary to a sequence to be detected in the target HPV nucleicacid, such that hybridization of the loop to its complementary sequencein the target forces disassociation of the stem, thereby distancing thefluorophore and quencher from each other. This results in unquenching ofthe fluorophore, causing an increase in fluorescence of the MB.

Details regarding standard methods of making and using MBs are wellestablished in the literature and MBs are available from a number ofcommercial reagent sources. Further details regarding methods of MBmanufacture and use are found, e.g., in Leone et al. (1995) “Molecularbeacon probes combined with amplification by NASBA enable homogenousreal-time detection of RNA,” Nucleic Acids Res. 26:2150-2155; Hsuih etal. (1997) “Novel, ligation-dependent PCR assay for detection ofhepatitis C in serum” J Clin Microbiol 34:501-507; Kostrikis et al.(1998) “Molecular beacons: spectral genotyping of human alleles” Science279:1228-1229; Sokol et al. (1998) “Real time detection of DNA:RNAhybridization in living cells” Proc. Natl. Acad. Sci. U.S.A.95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons forallele discrimination” Nature Biotechnology 16:49-53; Fang et al. (1999)“Designing a novel molecular beacon for surface-immobilized DNAhybridization studies” J. Am. Chem. Soc. 121:2921-2922; and Marras etal. (1999) “Multiplex detection of single-nucleotide variation usingmolecular beacons” Genet. Anal. Biomol. Eng. 14:151-156, all of whichare incorporated by reference. Aspects of MB construction and use arealso found in patent literature, such as U.S. Pat. No. 5,925,517 (Jul.20, 1999) to Tyagi et al. entitled “Detectably labeled dual conformationoligonucleotide probes, assays and kits;” U.S. Pat. No. 6,150,097 toTyagi et al (Nov. 21, 2000) entitled “Nucleic acid detection probeshaving non-FRET fluorescence quenching and kits and assays includingsuch probes” and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000),entitled “Wavelength-shifting probes and primers and their use in assaysand kits,” all of which are incorporated by reference.

MB components (e.g., oligos, including those labeled with fluorophoresor quenchers) can be synthesized using conventional methods. Some ofthese methods are described further above. For example, oligonucleotidesor peptide nucleic acids (PNAs) can be synthesized on commerciallyavailable automated oligonucleotide/PNA synthesis machines usingstandard methods. Labels can be attached to the oligonucleotides or PNAseither during automated synthesis or by post-synthetic reactions whichhave been described before see, e.g., Tyagi and Kramer (1996), supra.Aspects relating to the synthesis of functionalized oligonucleotides canalso be found in Nelson, et al. (1989) “Bifunctional OligonucleotideProbes Synthesized Using A Novel CPG Support Are Able To Detect SingleBase Pair Mutations” Nucleic Acids Res. 17:7187-7194, which isincorporated by reference. Labels/quenchers can be introduced to theoligonucleotides or PNAs, e.g., by using a controlled-pore glass columnto introduce, e.g., the quencher (e.g., a4-dimethylaminoazobenzene-4′-sulfonyl moiety (DABSYL). For example, thequencher can be added at the 3′ end of oligonucleotides during automatedsynthesis; a succinimidyl ester of 4-(4′-dimethylaminophenylazo)benzoicacid (DABCYL) can be used when the site of attachment is a primary aminogroup; and 4-dimethylaminophenylazophenyl-4′-maleimide (DABMI) can beused when the site of attachment is a sulphydryl group. Similarly,fluorescein can be introduced in the oligonucleotides, either using afluorescein phosphoramadite that replaces a nucleoside with fluorescein,or by using a fluorescein dT phosphoramadite that introduces afluorescein moiety at a thymidine ring via a linker. To link afluorescein moiety to a terminal location, iodoacetoamidofluorescein canbe coupled to a sulphydryl group. Tetrachlorofluorescein (TET) can beintroduced during automated synthesis using a 5′-tetrachloro-fluoresceinphosphoramadite. Other reactive fluorophore derivatives and theirrespective sites of attachment include the succinimidyl ester of5-carboxyrhodamine-6G (RHD) coupled to an amino group; an iodoacetamideof tetramethylrhodamine coupled to a sulphydryl group; an isothiocyanateof tetramethylrhodamine coupled to an amino group; or a sulfonylchlorideof Texas red coupled to a sulphydryl group. During the synthesis ofthese labeled components, conjugated oligonucleotides or PNAs can bepurified, if desired, e.g., by high pressure liquid chromatography orother methods.

A variety of commercial suppliers produce standard and custom molecularbeacons, including Cruachem (cruachem.com), Oswel Research Products Ltd.(UK; oswel.com), Research Genetics (a division of Invitrogen, HuntsvilleAla. (resgen.com)), the Midland Certified Reagent Company (Midland, Tex.mcrc.com) and Gorilla Genomics, LLC (Alameda, Calif.). A variety of kitswhich utilize molecular beacons are also commercially available, such asthe Sentinel™ Molecular Beacon Allelic Discrimination Kits fromStratagene (La Jolla, Calif.) and various kits from Eurogentec SA(Belgium, eurogentec.com) and Isogen Bioscience BV (The Netherlands,isogen.com).

In one embodiment, a real time PCR assay system that includes one ormore 5′-nuclease probes is used for detecting amplified HPV nucleicacids. These systems operate by using the endogenous endonucleaseactivity of certain polymerases to cleave a quencher or label free froman oligonucleotide of the invention that comprises the quencher andlabel, resulting in unquenching of the label. The polymerase onlycleaves the quencher or label upon initiation of replication, i.e., whenthe oligonucleotide is bound to the template and the polymerase extendsthe primer. Thus, an appropriately labeled oligonucleotide probe andpolymerase comprising the appropriate nuclease activity can be used todetect an HPV nucleic acid of interest. Real time PCR product analysisby, e.g., Fluorescent Resonance Energy Transfer (FRET) or the likeprovides a well-known technique for real time PCR monitoring that hasbeen used in a variety of contexts, which can be adapted for use withthe oligonucleotides and methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Laurendeau et al. (1999) “Quantitation of MYCgene expression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Clin Chem 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/ab1 fusiontranscripts” Cancer Research 59(13):3171-4, all of which areincorporated by reference).

X. Systems

The invention also provides a system for detecting HPV in a sample. Thesystem includes one or more oligonucleotides as described herein. Incertain embodiments, the oligonucleotides are arrayed on a solidsupport, whereas in others, they are provided in one or more containers,e.g., for assays performed in solution. The system also includes atleast one detector (e.g., a spectrometer, etc.) that detects bindingbetween nucleic acids and/or amplicons thereof from the sample and theoligonucleotides. Other detectors are described further below. Inaddition, the system also includes at least one controller operablyconnected to the detector. The controller includes one or moreinstructions sets that correlate the binding detected by the detectorwith a presence of HPV in the sample.

As referred to above, at least one container or solid support includesthe oligonucleotides in some embodiments of the invention. In theseembodiments, the system optionally further includes at least one thermalmodulator operably connected to the container or solid support tomodulate temperature in the container or on the solid support, and/or atleast one fluid transfer component (e.g., an automated pipettor, etc.)that transfers fluid to and/or from the container or solid support,e.g., for performing one or more nucleic acid amplification techniquesand/or nucleic acid hybridization assays in the container or on thesolid support.

Exemplary commercially available systems that are optionally utilized todetect HPV nucleic acids using the oligonucleotides described hereininclude, e.g., a COBAS TaqMan™ Analyzer or a COBAS AMPLICOR® Analyzerwhich are available from Roche Diagnostics Corporation (Indianapolis,Ind.), a LUMINEX 100™ system, which is available from the LuminexCorporation (Austin, Tex.), and the like.

The invention further provides a system that includes a computer orcomputer readable medium that includes a data set that comprises atleast one character string that corresponds to at least one sequenceselected from the group consisting of: SEQ ID NOS: 1-31 and complementsthereof. Typically, the system further includes an automatic synthesizercoupled to an output of the computer or computer readable medium. Theautomatic synthesizer accepts instructions from the computer or computerreadable medium, which instructions direct synthesis of, e.g., one ormore nucleic acids that correspond to one or more character strings inthe data set. Exemplary systems and system components are describedfurther below.

Detectors are structured to detect detectable signals produced, e.g., inor proximal to another component of the system (e.g., in container, on asolid support, etc.). Suitable signal detectors that are optionallyutilized, or adapted for use, in these systems detect, e.g.,fluorescence, phosphorescence, radioactivity, absorbance, refractiveindex, luminescence, or the like. Detectors optionally monitor one or aplurality of signals from upstream and/or downstream of the performanceof, e.g., a given assay step. For example, the detector optionallymonitors a plurality of optical signals, which correspond in position to“real time” results. Example detectors or sensors includephotomultiplier tubes, CCD arrays, optical sensors, temperature sensors,pressure sensors, pH sensors, conductivity sensors, scanning detectors,or the like. Each of these as well as other types of sensors isoptionally readily incorporated into the systems described herein.Optionally, the systems of the present invention include multipledetectors.

More specific exemplary detectors that are optionally utilized in thesesystems include, e.g., a resonance light scattering detector, anemission spectroscope, a fluorescence spectroscope, a phosphorescencespectroscope, a luminescence spectroscope, a spectrophotometer, aphotometer, and the like. Various synthetic components are alsoutilized, or adapted for, use in the systems of the invention including,e.g., automated nucleic acid synthesizers, e.g., for synthesizing theoligonucleotides described herein. Detectors and synthetic componentsthat are optionally included in the systems of the invention aredescribed further in, e.g., Skoog et al., Principles of InstrumentalAnalysis, 5^(th) Ed., Harcourt Brace College Publishers (1998) andCurrell, Analytical Instrumentation: Performance Characteristics andQuality, John Wiley & Sons, Inc. (2000), both of which are incorporatedby reference.

The systems of the invention also typically include controllers that areoperably connected to one or more components (e.g., detectors, syntheticcomponents, thermal modulator, fluid transfer components, etc.) of thesystem to control operation of the components. More specifically,controllers are generally included either as separate or integral systemcomponents that are utilized, e.g., to receive data from detectors, toeffect and/or regulate temperature in the containers, to effect and/orregulate fluid flow to or from selected containers, or the like.Controllers and/or other system components is/are optionally coupled toan appropriately programmed processor, computer, digital device, orother information appliance (e.g., including an analog to digital ordigital to analog converter as needed), which functions to instruct theoperation of these instruments in accordance with preprogrammed or userinput instructions, receive data and information from these instruments,and interpret, manipulate and report this information to the user.Suitable controllers are generally known in the art and are availablefrom various commercial sources.

Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display, etc.), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser. These components are illustrated further below.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of one or more controllers to carry out thedesired operation. The computer then receives the data from, e.g.,sensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as controlling fluid flow regulators in responseto fluid weight data received from weight scales or the like.

The computer can be, e.g., a PC (Intel x86 or Pentium chip-compatibleDOS™, OS2™, WINDOWS™, WINDOWS NT™, WINDOWS95™, WINDOWS98™, WINDOWS2000™,WINDOWS XP™, LINUX-based machine, a MACINTOSHT™, Power PC, or aUNIX-based (e.g., SUN™ work station) machine) or other commoncommercially available computer which is known to one of skill. Standarddesktop applications such as word processing software (e.g., MicrosoftWord™ or Corel WordPerfect™) and database software (e.g., spreadsheetsoftware such as Microsoft Excel™, Corel Quattro Pro™, or databaseprograms such as Microsoft Access™ or Paradox™) can be adapted to thepresent invention. Software for performing, e.g., controllingtemperature modulators and fluid flow regulators is optionallyconstructed by one of skill using a standard programming language suchas Visual basic, Fortran, Basic, Java, or the like.

FIGS. 1 and 2 are schematics showing representative example systems thatinclude logic devices in which various aspects of the present inventionmay be embodied. As will be understood by practitioners in the art fromthe teachings provided herein, the invention is optionally implementedin hardware and/or software. In some embodiments, different aspects ofthe invention are implemented in either client-side logic or server-sidelogic. As will be understood in the art, the invention or componentsthereof may be embodied in a media program component (e.g., a fixedmedia component) containing logic instructions and/or data that, whenloaded into an appropriately configured computing device, cause thatdevice to perform according to the invention. As will also be understoodin the art, a fixed media containing logic instructions may be deliveredto a viewer on a fixed media for physically loading into a viewer'scomputer or a fixed media containing logic instructions may reside on aremote server that a viewer accesses through a communication medium inorder to download a program component.

In particular, FIG. 1 schematically illustrate computer 100 to whichdetector 102 and fluid transfer component 104 are operably connected.Optionally, detector 102 and/or fluid transfer component 104 is operablyconnected to computer 100 via a server (not shown in FIG. 1). Duringoperation, fluid transfer component 104 typically transfers fluids, suchas sample aliquots comprising labeled HPV amplicons to oligonucleotidearray 106. Thereafter, detector 102 typically detects detectable signals(e.g., fluorescent emissions, etc.) produced by labeled amplicons thathybridize with oligonucleotides attached to oligonucleotide array 106after one or more washing steps are performed to wash awaynon-hybridized nucleic acids from oligonucleotide array 106 using fluidtransfer component 104. As additionally shown, thermal modulator 108 isalso operably connected to computer 100. Prior to performing ahybridization assay, target HPV nucleic acids can be amplified usinglabeled primer nucleic acids. The amplicons of these amplificationreactions are then typically transferred to oligonucleotide array 106using fluid transfer component 104, as described above, to perform thebinding assay. In some embodiments, binding assays are performedconcurrently with HPV nucleic acid amplification in thermal modulator108 using, e.g., molecular beacons, 5′-nuclease probes, or the like thatcomprise sequences selected from, e.g., SEQ ID NOS: 1-31. In theseembodiments, detector 102 detects detectable signals produced as theamplification reactions are performed using thermal modulator 108.

FIG. 2 schematically shows information appliance or digital device 200that may be understood as a logical apparatus that can read instructionsfrom media 202 and/or network port 204, which can optionally beconnected to server 206 having fixed media 208. Digital device 200 canthereafter use those instructions to direct server or client logic, asunderstood in the art, to embody aspects of the invention. One type oflogical apparatus that may embody the invention is a computer system asillustrated in 200, containing CPU 210, optional input devices 212 and214, disk drives 216 and optional monitor 218. Fixed media 202, or fixedmedia 208 over port 204, may be used to program such a system and mayrepresent a disk-type optical or magnetic media, magnetic tape, solidstate dynamic or static memory, or the like. In specific embodiments,the invention may be embodied in whole or in part as software recordedon this fixed media. Communication port 204 may also be used toinitially receive instructions that are used to program such a systemand may represent any type of communication connection. Optionally, theinvention is embodied in whole or in part within the circuitry of anapplication specific integrated circuit (ACIS) or a programmable logicdevice (PLD). In such a case, the invention may be embodied in acomputer understandable descriptor language, which may be used to createan ASIC, or PLD.

FIG. 2 also includes automatic synthesizer 220, which is operablyconnected to digital device 200 via server 206. Optionally, automaticsynthesizer 220 is directly connected to digital device 200. Duringoperation, automatic synthesizer 220 typically receives instructions tosynthesize one or more oligonucleotides that comprise a sequenceselected from, e.g., SEQ ID NOS: 1-31 or complements thereto, which areincluded in a data set comprised by, e.g., digital device 200 and/or acomputer readable medium, such as fixed media 202 and/or 208.

XI. Kits

The oligonucleotides employed in the methods of the present inventionare optionally packaged into kits. In addition, the kits may alsoinclude suitably packaged reagents and materials needed for DNAimmobilization, hybridization, and detection, such solid supports,buffers, enzymes, and DNA standards, as well as instructions forconducting the assay. Optionally, the oligonucleotides of the inventionare provided already attached or otherwise immobilized on solidsupports. As another option, oligonucleotides are provided free insolution in containers, e.g., for performing the detection methods ofthe invention in the solution phase. In some of these embodiments,oligonucleotides of the kits comprise labels and/or quencher moieties,such as when molecular beacons, 5′-nuclease probes, or the like comprisesequences selected from, e.g., SEQ ID NOS: 1-31. In certain embodiments,kits further include labeled primers for amplifying target HPV sequencesin a sample.

The kits also typically include one or more of: a set of instructionsfor contacting the oligonucleotides with nucleic acids from a sample oramplicons thereof and detecting binding between the oligonucleotides andHPV nucleic acids, if any, or at least one container for packaging theoligonucleotides and the set of instructions. Exemplary solid supportsinclude in the kits of the invention are optionally selected from, e.g.,a plate, a bead, a microwell plate, a microbead, a fiber, a whisker, acomb, a hybridization chip, a membrane, a single crystal, a ceramiclayer, a self-assembling monolayer, or the like.

In some embodiments, the kit further includes at least one primernucleic acid that is at least partially complementary to at least onesegment of the L1 region of HPV, e.g., for amplifying a segment of thatregion of the HPV genome. In these embodiments, the kit typicallyfurther includes a set of instructions for amplifying one or moresubsequences of the L1 region of HPV with the primer nucleic acids, atleast one nucleotide incorporating biocatalyst, and one or morenucleotides. In certain embodiments, the primer nucleic acids compriseat least one label (e.g., a fluorescent dye, a radioisotope, etc.).Suitable labels are described further herein. For example, the primernucleic acid is optionally conjugated with biotin or a biotinderivative. In these embodiments, the kit typically further includes anenzyme conjugated with avidin or an avidin derivative, or streptavidinor a streptavidin derivative, e.g., for effecting the detection ofbinding between the oligonucleotides of the invention and target nucleicacids. In these embodiments, the kit generally further includes at leastone nucleotide incorporating biocatalyst (e.g., a polymerase, a ligase,or the like). In these embodiments, the kit typically also furthercomprising one or more nucleotides, e.g., for use in amplifying thetarget nucleic acids. Optionally, at least one of the nucleotidescomprises a label. In some of these embodiments, the kits furtherinclude at least one pyrophosphatase (e.g., a thermostablepyrophosphatase), e.g., for use in minimizing pyrophosphorolysis, uracilN-glycosylase (UNG), e.g., for use in applications where protectionagainst carry-over contamination is desirable.

XII. Examples Performance Data for Cross-Reactive Oligonucleotides

It is understood that the examples and embodiments described herein arefor illustrative purposes only and are not intended to limit the scopeof the claimed invention. It is also understood that variousmodifications or changes in light the examples and embodiments describedherein will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

These examples show certain performance data for various HPVoligonucleotides of the present invention, which were utilized as probesin this example. More specifically, probe binding specificity fordifferent high- and low-risk HPV targets as well as probe meltingcharacteristics are illustrated in these examples.

Example 1 Probe Binding Specificity

Certain oligonucleotides were used as probes to test for the probebinding specificity of each oligonucleotide against or relative tovarious high- and low-risk HPV targets.

A. Materials & Methods

The specific oligonucleotides utilized as probes in these assays wereA3X5XA (SEQ ID NO: 12), A3X5XC (SEQ ID NO: 15), and A3X5XD (SEQ ID NO:16). A3X5XA is an unmodified oligonucleotide, while A3X5XC contains fivemodified nucleotide substitutions, namely, four 5-propynyl-dUsubstitutions and one 5-methyl-dC substitution. The A3X5XDoligonucleotide also includes five modified nucleotide substitutions atthe same nucleotide positions as the A3X5XC oligonucleotide, but thesemodified nucleotides have different chemistries (i.e., four 2′-O-methylRibo-U substitutions and one 2′-O-methyl Ribo-C substitution).

Using a Sodium Phosphate/EDTA coat buffer solution mixed withapproximately 100 picomoles of each desired probe per ml, microwellplates (MWP) were coated by pipetting 100 μl of probe solution into eachwell (resulting in a concentration of 10 pmoles/well), sealing withplastic, and incubating overnight at room temperature. Each plate wasthen washed with a buffer solution of PBS+EDTA, dried on the lab bench,bagged in plastic with dessicant, and stored at 4 C.

Next, HPV plasmids were amplified in a PCR master mix including 10 mMTris/50 mM KCl at pH8.3, 7.5 Units per reaction AmpliTaq Gold, 200 μMeach of dATP, dCTP, dGTP, 400 uM dUTP, 5 μM each of upstream anddownstream primers, 1 unit of UNG per reaction, 0.05% Sodium azide, 3.75mM Magnesium Chloride, & 0.025% Tween-20 in a 100 μL reaction volume.The copy input for the low-risk genotypes indicated was 10⁷ copies perPCR, whereas that for the high-risk genotypes denoted was 10⁴ copies perPCR. The thermal cycling profile used on the PE 9700 was:

HOLD Program: 2 min 50° C.

HOLD Program: 9 min 95° C.

CYCLE Program (10 Cycles): 30 sec 95° C., 30 sec 48° C., 30 sec 72° C.

CYCLE Program (30 Cycles): 30 sec 95° C., 45 sec 54° C., 30 sec 72° C.

HOLD Program: 72° C. Indefinitely

The ramp rate was set to 50%, the “Ramp Speed” was at “Max”, and the“Reaction Volume” was “100 μL”.

Following PCR amplification, the HPV amplicon was chemically denaturedwith 100 μL Denaturation Solution (1.6% (w/w) Sodium hydroxide, EDTA,Thymol blue) to form single-stranded DNA.

100 μL of HPV Hybridization Buffer (solution containing Sodium phosphatebuffer, <25% Sodium thiocyanate and <0.2% solubilizer) was added to eachwell on the MWP. Then, 25 μL of denatured amplicon was added to thewells. The biotin-labeled HPV amplicon was hybridized to theoligonucleotide probes during incubation for 1 hour at 37° C. Followingthe hybridization, the MWP was washed with 1× Phosphate/Sodiumchloride/EDTA buffer to remove unbound material. Avidin-HorseradishPeroxidase Conjugate was then added to each well of the MWP andincubated. The MWP was washed again to remove unbound conjugate and asubstrate solution containing hydrogen peroxide and tetramethylbenzidine(TMB) was added to the wells.

The reaction was stopped by addition of a weak acid and the absorbanceat 450 nm was measured using an automated microwell plate reader. Theseabsorbance values were represented as bar graphs to show the relative ODreadings obtained with the plate reader.

B. Results

FIG. 3 is a graph that illustrates the specificity of several probes forvarious HPV targets. In particular, the abscissa of the graph representsthe absorbance for each polymerase chain reaction mixture amplicon,hybridized to the immobilized probe, subjected to an enzymatic colorprecipitation reaction and then measured at 450 nm, while the ordinaterepresents the genotypes of the particular HPV targets assayed. Thesespecificity experiments have also been repeated using the A3X5XColigonucleotide in a pool of several oligonucleotides specific to highrisk HPV genotypes as well (data not shown).

Example 2 Probe Melting Characteristics

The unmodified A3X5XA oligonucleotide was compared with the modifiedA3X5XC oligonucleotide to examine the difference in melting temperature.

A. Materials & Methods

The oligonucleotides were each assayed at 0.1 micromolar (1 μM) in abuffer containing 120 mM potassium acetate pH 8.3, 50 mM tricine pH 8.3,4.5% (w/v) glycerol, 200 μM each of dATP, dCTP, dGTP, 400 μM dUTP, 4 mMmagnesium acetate pH 6.5, 0.4×SYBR® Green (from 10,000× stock), and 0.2μM of complementary strand oligonucleotide.

The DNA strands in the buffer were first annealed by raising thetemperature to 100 degrees C. and then lowering it to 35 degrees C. at 2degrees/second. After annealing, the temperature was raised from 35degrees C. to 70 degrees C. at 2 degrees/second to dissociate (melt) theDNA duplex. The change of state from double-strand to single-strand (DNAdissociation) as the temperature is raised results in a reduction of dyebinding (SYBR® Green) to the DNA, and to a concomitant decrease influorescence. This change in fluorescent emission with temperature wasmeasured in an ABI Prism® 7000 Sequence Detection System (AppliedBiosystems, Foster City, Calif., USA) using the FAM filter (approximatemaximum wavelength 515 nanometers). The derivatives of the resultantfluorescence readings were graphed versus temperature to more easilyvisualize the inflection point at which 50% of the duplex dissociated(T_(m)).

B. Results

FIG. 4 is a graph that shows melting curves for the A3X5XA and A3X5XColigonucleotides using a complementary sequence with no mismatches(i.e., antiA3X5XA). The abscissa of the graph represents the derivativeof the fluorescence, while the ordinate represents the temperature. Asshown, a melting temperature increase of about 4.5° C. was detected forthe A3X5XC oligonucleotide relative to the A3X5XA oligonucleotide usingthe perfect match complementary oligonucleotide.

FIG. 5 is a graph that shows melting curves for the A3X5XA and A3X5XColigonucleotides using two target sequences, anti33A3X5X andanti52A3X5X, that each included a single distinct mismatch to theoligonucleotides. The abscissa of the graph represents the derivative ofthe fluorescence, while the ordinate represents the temperature. Asshown, the difference in melting temperature between the A3X5XAoligonucleotide and the A3X5XC oligonucleotide detected for targetsequences was approximately 2.5° C.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. An oligonucleotide comprising a nucleic acid sequence having at least95% sequence identity to one of SEQ ID NOS: 4 and 5, or a complement ofone of SEQ ID NOS: 4 and 5, which oligonucleotide consists of 100 orfewer nucleotides.
 2. The oligonucleotide of claim 1, further comprisingat least one labeling moiety and/or at least one quencher moiety.
 3. Theoligonucleotide of claim 1, wherein the nucleic acid comprises at leastone modified nucleotide substitution.
 4. The oligonucleotide of claim 3,wherein the modified nucleotide is selected from the group consistingof: a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a 7-deaza-dG, an N4-ethyl-dC,and an N6-methyl-dA.
 5. A method of determining a presence of at leastone high-risk human papillomavirus (HPV) type in a sample, the methodcomprising: (a) contacting nucleic acids and/or amplicons thereof fromthe sample with at least one oligonucleotide that comprises a nucleicacid sequence having at least 95% sequence identity to one of SEQ IDNOS: 4 and 5, or a complement of one of SEQ ID NOS: 4 and 5, whicholigonucleotide consists of 100 or fewer nucleotides; and, (b)monitoring binding between the nucleic acids and/or amplicons thereof,and the oligonucleotide, wherein detectable binding between the nucleicacids and/or amplicons thereof, and the oligonucleotide, determines thepresence of the high-risk HPV type in the sample.
 6. The method of claim5, wherein the high-risk HPV type comprises one or more of HPV31, HPV33,HPV35, HPV52, HPV56, or HPV58.
 7. An oligonucleotide comprising anucleic acid sequence having 100% sequence identity to SEQ ID NO: 6 or acomplement of SEQ ID NO: 6, which oligonucleotide consists of 100 orfewer nucleotides.
 8. The oligonucleotide of claim 7, further comprisingat least one labeling moiety and/or at least one quencher moiety.
 9. Theoligonucleotide of claim 7, wherein the nucleic acid comprises at leastone modified nucleotide substitution.
 10. The oligonucleotide of claim9, wherein the modified nucleotide is selected from the group consistingof: a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA,a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, aC7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-0-methyl Ribo-U, 2′-0-methyl Ribo-C,an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a 7-deaza-dG, an N4-ethyl-dC,and an N6-methyl-dA.
 11. A method of determining a presence of at leastone high-risk human papillomavirus (HPV) type in a sample, the methodcomprising: (a) contacting nucleic acids and/or amplicons thereof fromthe sample with at least one oligonucleotide that comprises a nucleicacid sequence having 100% sequence identity to SEQ ID NO: 6 or acomplement of SEQ ID NO: 6, which oligonucleotide consists of 100 orfewer nucleotides; and, (b) monitoring binding between the nucleic acidsand/or amplicons thereof, and the oligonucleotide, wherein detectablebinding between the nucleic acids and/or amplicons thereof, and theoligonucleotide, determines the presence of the high-risk HPV type inthe sample.
 12. The method of claim 11, wherein the high-risk HPV typecomprises one or more of: HPV31, HPV33, HPV35, HPV52, HPV56, or HPV58.